Nucleophilic catalysts for oxime linkage

ABSTRACT

The invention relates to materials and methods of conjugating a water soluble polymer to an oxidized carbohydrate moiety of a therapeutic protein comprising contacting the oxidized carbohydrate moiety with an activated water soluble polymer under conditions that allow conjugation. More specifically, the present invention relates to the aforementioned materials and methods wherein the water soluble polymer contains an active aminooxy group and wherein an oxime or hydrazone linkage is formed between the oxidized carbohydrate moiety and the active aminooxy group on the water soluble polymer, and wherein the conjugation is carried out in the presence of a nucleophilic catalyst.

FIELD OF THE INVENTION

The present invention relates to materials and methods for conjugating awater soluble polymer to a protein.

BACKGROUND OF THE INVENTION

The preparation of conjugates by forming a covalent linkage between thewater soluble polymer and the therapeutic protein can be carried out bya variety of chemical methods. PEGylation of polypeptide drugs protectsthem in circulation and improves their pharmacodynamic andpharmacokinetic profiles (Harris and Chess, Nat Rev Drug Discov. 2003;2:214-21). The PEGylation process attaches repeating units of ethyleneglycol (polyethylene glycol (PEG)) to a polypeptide drug. PEG moleculeshave a large hydrodynamic volume (5-10 times the size of globularproteins), are highly water soluble and hydrated, non-toxic,non-immunogenic and rapidly cleared from the body. PEGylation ofmolecules can lead to increased resistance of drugs to enzymaticdegradation, increased half-life in vivo, reduced dosing frequency,decreased immunogenicity, increased physical and thermal stability,increased solubility, increased liquid stability, and reducedaggregation. The first PEGylated drugs were approved by the FDA in theearly 1990s. Since then, the FDA has approved several PEGylated drugsfor oral, injectable, and topical administration.

Polysialic acid (PSA), also referred to as colominic acid (CA), is anaturally occurring polysaccharide. It is a homopolymer ofN-acetylneuraminic acid with α(2→8) ketosidic linkage and containsvicinal diol groups at its non-reducing end. It is negatively chargedand a natural constituent of the human body. It can easily be producedfrom bacteria in large quantities and with pre-determined physicalcharacteristics (U.S. Pat. No. 5,846,951). Because thebacterially-produced PSA is chemically and immunologically identical toPSA produced in the human body, bacterial PSA is non-immunogenic, evenwhen coupled to proteins. Unlike some polymers, PSA acid isbiodegradable. Covalent coupling of colominic acid to catalase andasparaginase has been shown to increase enzyme stability in the presenceof proteolytic enzymes or blood plasma. Comparative studies in vivo withpolysialylated and unmodified asparaginase revealed that polysialylationincreased the half-life of the enzyme (Fernandes and Gregoriadis, Int JPharm. 2001; 217:215-24).

Coupling of PEG-derivatives to peptides or proteins is reviewed byRoberts et al. (Adv Drug Deliv Rev 2002; 54:459-76). One approach forcoupling water soluble polymers to therapeutic proteins is theconjugation of the polymers via the carbohydrate moieties of theprotein. Vicinal hydroxyl (OH) groups of carbohydrates in proteins canbe easily oxidized with sodium periodate (NaIO₄) to form active aldehydegroups (Rothfus et Smith, J Biol Chem 1963; 238:1402-10; van Lenten etAshwell, J Biol Chem 1971; 246:1889-94). Subsequently the polymer can becoupled to the aldehyde groups of the carbohydrate by use of reagentscontaining, for example, an active hydrazide group (Wilchek M and BayerE A, Methods Enzymol 1987; 138:429-42). A more recent technology is theuse of reagents containing aminooxy groups which react with aldehydes toform oxime linkages (WO 96/40662, WO2008/025856).

Additional examples describing conjugation of a water soluble polymer toa therapeutic protein are described in WO 06/071801 which teaches theoxidation of carbohydrate moieties in Von Willebrand factor andsubsequent coupling to PEG using hydrazide chemistry; US Publication No.2009/0076237 which teaches the oxidation of rFVIII and subsequentcoupling to PEG and other water soluble polymers (e.g. PSA, HES,dextran) using hydrazide chemistry; WO 2008/025856 which teachesoxidation of different coagulation factors, e.g. rFIX, FVIII and FVIIaand subsequent coupling to e.g., PEG, using aminooxy chemistry byforming an oxime linkage; and U.S. Pat. No. 5,621,039 which teaches theoxidation of FIX and subsequent coupling to PEG using hydrazidechemistry.

Recently, an improved method was described comprising mild periodateoxidation of sialic acids to generate aldehydes followed by reactionwith an aminooxy group containing reagent in the presence of catalyticamounts of aniline (Dirksen A., and Dawson P E, Bioconjugate Chem. 2008;19, 2543-8; and Zeng Y et al., Nature Methods 2009; 6:207-9). Theaniline catalysis dramatically accelerates the oxime ligation, allowingthe use of very low concentrations of the reagent. The use ofnucleophilic catalysts are also described in Dirksen, A., et al., J AmChem Soc., 128:15602-3 (2006); Dirksen, A., et al., Angew chem. Int Ed.,45:7581-4 (2006); Kohler, J. J., ChemBioChem., 10:2147-50 (2009);Giuseppone, N., et al., J Am Chem Soc., 127:5528-39 (2005); andThygesen, M. B., et al., J Org Chem., 75:1752-5 (2010).

Although aniline catalysis can accelerate the oxime ligation allowingshort reaction times and the use of low concentrations of the aminooxyreagent, aniline has toxic properties that must be considered when, forexample, the conjugated therapeutic protein to form the basis of apharmaceutical. For example, aniline has been shown to inducemethemoglobinemia (Harrison, J. H., and Jollow, D. J., MolecularPharmacology, 32(3) 423-431, 1987). Long-term dietary treatment of ratshas been shown to induce tumors in the spleen (Goodman, D G., et al., JNatl Cancer Inst., 73(1):265-73, 1984). In vitro studies have also shownthat aniline has the potential to induce chromosome mutations and hasthe potentially genotoxic activity (Bombhard E. M. et Herbold B,Critical Reviews in Toxicology 35, 783-835, 2005).

There remains a need to develop materials and methods for conjugatingwater soluble polymers to proteins that improves the protein'spharmacodynamic and/or pharmacokinetic properties while minimizing thecosts associated with the various reagents and minimizing the healthrisks to the patient recipient.

SUMMARY OF THE INVENTION

The present invention provides materials and methods for conjugatingpolymers to proteins that improves the protein's pharmacodynamic and/orpharmacokinetic properties while minimizing the costs associated withthe various reagents and the health risks to the patient recipients whenthe conjugation reaction is catalyzed by a nucleophilic catalyst. Invarious embodiments of the invention, alternative catalysts tosubstitute for aniline are provided.

The present disclosure also provides an optimized procedure forpreparation of various water-soluble polymer-aminooxy linker reagentsthat can be used to conjugate therapeutic proteins as described herein.Recent NMR studies showed that side reactions at the reducing end of PSAcan occur if the preparation of the PSA-aminooxy reagent is performed atroom temperature. Thus, in various embodiments of the presentdisclosure, the new process is carried out at a temperature between 2-8°C. In one embodiment, the PSA-aminooxy reagent is prepared at 4° C.according to the process described herein. In addition, purification ofthe reagent using a chromatographic purification step (e.g., IEX) at atemperature between 2-8° C. is also contemplated by the presentdisclosure. Unwanted side reactions at the reducing end of the PSA aresubstantially reduced when the entire process (chemical reaction andpurification of the conjugate by IEX) is performed at a temperaturebetween 2-8° C.

In one embodiment according to the present disclosure, a method ofconjugating a water soluble polymer to an oxidized carbohydrate moietyof a therapeutic protein is provided comprising contacting the oxidizedcarbohydrate moiety with an activated water soluble polymer underconditions that allow conjugation; said water soluble polymer containingan active aminooxy group and is selected from the group consisting ofpolyethylene glycol (PEG), branched PEG, PolyPEG® (Warwick EffectPolymers; Coventry, UK), polysialic acid (PSA), starch, hydroxyalkylstarch (HAS), hydroxylethyl starch (HES), carbohydrate, polysaccharides,pullulane, chitosan, hyaluronic acid, chondroitin sulfate, dermatansulfate, starch, dextran, carboxymethyl-dextran, polyalkylene oxide(PAO), polyalkylene glycol (PAG), polypropylene glycol (PPG),polyoxazoline, polyacryloylmorpholine, polyvinyl alcohol (PVA),polycarboxylate, polyvinylpyrrolidone, polyphosphazene, polyoxazoline,polyethylene-co-maleic acid anhydride, polystyrene-co-maleic acidanhydride, poly(1-hydroxymethylethylene hydroxymethylformal) (PHF),2-methacryloyloxy-2′-ethyltrimethylammoniumphosphate (MPC); wherein thewater soluble polymer containing an active aminooxy group is prepared bya method comprising: a) incubating a solution comprising an oxidizedwater-soluble polymer with an aminooxy linker comprising an activeaminooxy group under conditions that allow the formation of a stableoxime linkage between the oxidized water-soluble polymer and theactivated aminooxy linker, said conditions comprising a time periodbetween about 1 minute and about 24 hours; a temperature between about2° C. and about 8° C.; in the presence or absence of light, and with orwithout stirring; thereby forming a water-soluble polymer containing anactive aminooxy group; and b) purifying the water soluble polymercontaining an active aminooxy group by a method selected from the groupconsisting of chromatography, filtration, dialysis, and precipitation,at a temperature between about 2° C. and about 8° C.; said carbohydratemoiety oxidized by incubation with a buffer comprising an oxidizingagent selected from the group consisting of sodium periodate (NaIO₄),lead tetraacetate (Pb(OAc)₄) and potassium perruthenate (KRuO4); whereinan oxime linkage is formed between the oxidized carbohydrate moiety andthe active aminooxy group on the water soluble polymer; and wherein saidoxime linkage formation is catalyzed by a nucleophilic catalyst selectedfrom the group consisting of o-amino benzoic acid, m-amino benzoic acid,p-amino benzoic acid, sulfanilic acid, o-aminobenzamide, o-toluidine,m-toluidine, p-toluidine, o-anisidine, m-anisidine, and p-anisidine.

In another embodiment, the aforementioned method is provided wherein thesolution comprising the oxidized water-soluble polymer and the aminooxylinker comprising an active aminooxy group is incubated at 4° C. for 1 hin the absence of light with stirring. In another embodiment, theaforementioned method is provided wherein the water soluble polymercontaining an active aminooxy group is purified by anion exchangechromatography at a temperature of 4° C. In another embodiment, anaforementioned method is provided wherein the oxidized water solublepolymer is PSA and is oxidized by incubation with NaIO₄.

In another embodiment of the present disclosure, a method of conjugatinga water soluble polymer to an oxidized carbohydrate moiety of atherapeutic protein is provided comprising contacting the oxidizedcarbohydrate moiety with an activated water soluble polymer underconditions that allow conjugation; said water soluble polymer containingan active aminooxy group and is selected from the group consisting ofpolyethylene glycol (PEG), branched PEG, PolyPEG® (Warwick EffectPolymers; Coventry, UK), polysialic acid (PSA), starch, hydroxyalkylstarch (HAS), hydroxylethyl starch (HES), carbohydrate, polysaccharides,pullulane, chitosan, hyaluronic acid, chondroitin sulfate, dermatansulfate, starch, dextran, carboxymethyl-dextran, polyalkylene oxide(PAO), polyalkylene glycol (PAG), polypropylene glycol (PPG),polyoxazoline, polyacryloylmorpholine, polyvinyl alcohol (PVA),polycarboxylate, polyvinylpyrrolidone, polyphosphazene, polyoxazoline,polyethylene-co-maleic acid anhydride, polystyrene-co-maleic acidanhydride, poly(1-hydroxymethylethylene hydroxymethylformal) (PHF),2-methacryloyloxy-2′-ethyltrimethylammoniumphosphate (MPC); wherein thewater soluble polymer containing an active aminooxy group is prepared bya method comprising: a) incubating a solution comprising a water-solublepolymer with an aminooxy linker comprising an active aminooxy groupunder conditions that allow the formation of a stable oxime linkagebetween the water-soluble polymer and the activated aminooxy linker,said conditions comprising a time period between about 1 minute andabout 24 hours; a temperature between about 22° C. and about 37° C.; inthe presence or absence of light, and with or without stirring; therebyforming a water-soluble polymer containing an active aminooxy group; andb) purifying the water soluble polymer containing an active aminooxygroup by a method selected from the group consisting of chromatography,filtration, dialysis, and precipitation; said carbohydrate moietyoxidized by incubation with a buffer comprising an oxidizing agentselected from the group consisting of sodium periodate (NaIO₄), leadtetraacetate (Pb(OAc)₄) and potassium perruthenate (KRuO4); wherein anoxime linkage is formed between the oxidized carbohydrate moiety and theactive aminooxy group on the water soluble polymer; and wherein saidoxime linkage formation is catalyzed by a nucleophilic catalyst selectedfrom the group consisting of, o-amino benzoic acid, m-amino benzoicacid, p-amino benzoic acid, sulfanilic acid, o-aminobenzamide,o-toluidine, m-toluidine, p-toluidine, o-anisidine, m-anisidine, andp-anisidine.

In another embodiment, the aforementioned method is provided wherein thesolution comprising the water-soluble polymer and the aminooxy linkercomprising an active aminooxy group is incubated at 22° C. for 2 h inthe absence of light with stirring. In another embodiment, theaforementioned method is provided wherein the solution comprising thewater-soluble polymer and the aminooxy linker comprising an activeaminooxy group is incubated at 22° C. for 2 h in the absence of lightwith stirring; said method further comprising the step of increasing thetemperature of the solution to a temperature between about 32° C. andabout 37° C. and incubating for an additional 12-24 hours. In anotherembodiment, the aforementioned method is provided comprising theadditional step of adding an additional amount of aminooxy linkercomprising an active aminooxy group immediately prior to increasing thetemperature. In another embodiment, the aforementioned method isprovided wherein the water soluble polymer containing an active aminooxygroup is purified by a method selected from the group consisting ofdialysis, ultrafiltration/diafiltration (UF/DF), and chromatography at atemperature of 22° C. In another embodiment, the aforementioned methodis provided further comprising the step of purifying the water solublepolymer containing an active aminooxy group by a method selected fromthe group consisting of dialysis, UF/DF or chromatography at 4° C.

In various embodiments of the present disclosure, an aforementionedmethod is provided wherein the therapeutic protein selected from thegroup consisting of Factor IX (FIX), Factor VIII (FVIII), Factor VIIa(FVIIa), Von Willebrand Factor (VWF), Factor FV (FV), Factor X (FX),Factor XI (FXI), Factor XII (FXII), thrombin (FII), protein C, proteinS, tPA, PAI-1, tissue factor (TF), ADAMTS 13 protease, IL-1 alpha, IL-1beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-11, colony stimulating factor-1(CSF-1), M-CSF, SCF, GM-CSF, granulocyte colony stimulating factor(G-CSF), EPO, interferon-alpha (IFN-alpha), consensus interferon,IFN-beta, IFN-gamma, IFN-omega, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13,IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23,IL-24, IL-31, IL-32 alpha, IL-33, thrombopoietin (TPO), Ang-1, Ang-2,Ang-4, Ang-Y, angiopoietin-like polypeptide 1 (ANGPTL1),angiopoietin-like polypeptide 2 (ANGPTL2), angiopoietin-like polypeptide3 (ANGPTL3), angiopoietin-like polypeptide 4 (ANGPTL4),angiopoietin-like polypeptide 5 (ANGPTL5), angiopoietin-like polypeptide6 (ANGPTL6), angiopoietin-like polypeptide 7 (ANGPTL7), vitronectin,vascular endothelial growth factor (VEGF), angiogenin, activin A,activin B, activin C, bone morphogenic protein-1, bone morphogenicprotein-2, bone morphogenic protein-3, bone morphogenic protein-4, bonemorphogenic protein-5, bone morphogenic protein-6, bone morphogenicprotein-7, bone morphogenic protein-8, bone morphogenic protein-9, bonemorphogenic protein-10, bone morphogenic protein-11, bone morphogenicprotein-12, bone morphogenic protein-13, bone morphogenic protein-14,bone morphogenic protein-15, bone morphogenic protein receptor IA, bonemorphogenic protein receptor IB, bone morphogenic protein receptor II,brain derived neurotrophic factor, cardiotrophin-1, ciliary neutrophicfactor, ciliary neutrophic factor receptor, cripto, cryptic,cytokine-induced neutrophil chemotactic factor 1, cytokine-inducedneutrophil, chemotactic factor 2α, cytokine-induced neutrophilchemotactic factor 2β, β endothelial cell growth factor, endothelin 1,epidermal growth factor, epigen, epiregulin, epithelial-derivedneutrophil attractant, fibroblast growth factor 4, fibroblast growthfactor 5, fibroblast growth factor 6, fibroblast growth factor 7,fibroblast growth factor 8, fibroblast growth factor 8b, fibroblastgrowth factor 8c, fibroblast growth factor 9, fibroblast growth factor10, fibroblast growth factor 11, fibroblast growth factor 12, fibroblastgrowth factor 13, fibroblast growth factor 16, fibroblast growth factor17, fibroblast growth factor 19, fibroblast growth factor 20, fibroblastgrowth factor 21, fibroblast growth factor acidic, fibroblast growthfactor basic, glial cell line-derived neutrophic factor receptor α1,glial cell line-derived neutrophic factor receptor α2, growth relatedprotein, growth related protein α, growth related protein β, growthrelated protein γ, heparin binding epidermal growth factor, hepatocytegrowth factor, hepatocyte growth factor receptor, hepatoma-derivedgrowth factor, insulin-like growth factor I, insulin-like growth factorreceptor, insulin-like growth factor II, insulin-like growth factorbinding protein, keratinocyte growth factor, leukemia inhibitory factor,leukemia inhibitory factor receptor α, nerve growth factor nerve growthfactor receptor, neuropoietin, neurotrophin-3, neurotrophin-4,oncostatin M (OSM), placenta growth factor, placenta growth factor 2,platelet-derived endothelial cell growth factor, platelet derived growthfactor, platelet derived growth factor A chain, platelet derived growthfactor AA, platelet derived growth factor AB, platelet derived growthfactor B chain, platelet derived growth factor BB, platelet derivedgrowth factor receptor α, platelet derived growth factor receptor β,pre-B cell growth stimulating factor, stem cell factor (SCF), stem cellfactor receptor, TNF, TNF0, TNF1, TNF2, transforming growth factor α,transforming growth factor β, transforming growth factor β1,transforming growth factor β1.2, transforming growth factor β2,transforming growth factor β3, transforming growth factor β5, latenttransforming growth factor β1, transforming growth factor β bindingprotein I, transforming growth factor β binding protein II, transforminggrowth factor β binding protein III, thymic stromal lymphopoietin(TSLP), tumor necrosis factor receptor type I, tumor necrosis factorreceptor type II, urokinase-type plasminogen activator receptor,phospholipase-activating protein (PUP), insulin, lectin ricin,prolactin, chorionic gonadotropin, follicle-stimulating hormone,thyroid-stimulating hormone, tissue plasminogen activator, IgG, IgE,IgM, IgA, and IgD, α-galactosidase, β-galactosidase, DNAse, fetuin,leutinizing hormone, estrogen, insulin, albumin, lipoproteins,fetoprotein, transferrin, thrombopoietin, urokinase, integrin, thrombin,leptin, Humira (adalimumab), Prolia (denosumab), Enbrel (etanercept), aprotein in Table 1, or a biologically active fragment, derivative orvariant thereof.

In still another embodiment, the aforementioned method is providedwherein a solution comprising an initial concentration of thetherapeutic protein between about 0.3 mg/ml and about 3.0 mg/ml isadjusted to a pH value between about 5.0 and about 8.0 prior tocontacting with the activated water soluble polymer. In one embodiment,the initial concentration of the therapeutic protein is about 1.0 mg/mland the pH is about 6.0.

In another embodiment, the aforementioned method is provided wherein thetherapeutic protein is contacted by a desired excess concentration ofactivated water soluble polymer, wherein the excess concentration isbetween about 1-molar and about 300-molar excess. In one embodiment, theexcess concentration is about 50-fold molar excess.

In another embodiment, the aforementioned method is provided wherein thetherapeutic protein is incubated with the activated water solublepolymer under conditions comprising a time period between about 0.5hours and about 24 hours; a temperature between about 2° C. and about37° C.; in the presence or absence of light; and with or withoutstirring. In one embodiment, the conditions comprise a time period ofabout 120 minutes, a temperature of about 22° C., the absence of light;and with stirring.

In yet another embodiment, an aforementioned method is provided whereinthe nucleophilic catalyst is added in an amount to result in a finalconcentration between about 1.0 mM and about 50 mM nucleophiliccatalyst, under conditions comprising a time period between about 0.1minutes and about 30 minutes; a temperature between about 2° C. andabout 37° C.; in the presence or absence of light; and with or withoutstirring. In one embodiment, the final concentration of the nucleophiliccatalyst is about 10 mM, and the conditions comprise a time period of upto about 15 minutes, a temperature of about 22° C., the absence oflight; and with stirring.

In yet another embodiment, the aforementioned method is provided whereinthe oxidizing agent is added in an amount to result in a finalconcentration between about 50 μM and about 1000 μM oxidizing agent,under conditions comprising a time period between about 0.1 minutes and120 minutes; a temperature between about 2° C. and about 37° C.; in thepresence or absence of light; and with or without stirring. In oneembodiment, the final concentration of oxidizing agent is about 400 μM,and the conditions comprise a time period of about 10 minutes, atemperature of about 22° C., the absence of light and with stirring.

In still another embodiment, the aforementioned method is providedwherein the conjugating the water soluble polymer to the oxidizedcarbohydrate moiety of the therapeutic protein is stopped by theaddition of a quenching agent selected from the group consisting ofL-cysteine, methionine, glutathione, glycerol, sodium meta bisulfite(Na₂S₂O₅), tryptophane, tyrosine, histidine or derivatives thereof,kresol, imidazol, and combinations thereof; wherein the quenching agentis added in an amount to result in a final concentration between about 1mM and about 100 mM quenching agent, under conditions comprising a timeperiod between about 5 minutes and about 120 minutes; a temperaturebetween about 2° C. and about 37° C.; in the presence or absence oflight; and with or without stirring. In one embodiment the quenchingagent is L-cysteine. In still another embodiment, the L-cysteine isadded to result in a final concentration of about 10 mM and theconditions comprise a time period of about 60 minutes, a temperature ofabout 22° C., the absence of light and with stirring.

In another embodiment, an aforementioned method is provided comprising:a) a first step comprising adjusting the pH value of a solutioncomprising the therapeutic protein to a pH value between about 5.0 andabout 8.0, wherein the therapeutic protein concentration is betweenabout 0.3 mg/ml and about 3.0 mg/ml; b) a second step comprisingoxidizing one or more carbohydrates on the therapeutic protein, whereinthe oxidizing agent is added to the solution in the first step to resultin a final concentration between about 50 μM and about 1000 μM, underconditions comprising a time period between about 0.1 minutes and about120 minutes; a temperature between about 2° C. and about 37° C.; in thepresence or absence of light, and with or without stirring; c) a thirdstep comprising contacting the therapeutic protein with a desired excessconcentration of activated water soluble polymer, wherein the excessconcentration is between about 1-molar excess and about 300-molarexcess, under conditions comprising a time period between about 0.5hours and about 24 hours, a temperature between about 2° C. and about37° C.; in the presence or absence of light; and with or withoutstirring; d) a fourth step comprising adding a nucleophilic catalyst tothe solution of the third step, wherein the nucleophilic catalyst isadded to result in a final concentration between about 1 mM and about 50mM, under conditions comprising a time period between about 0.1 minutesand about 30 minutes; a temperature between about 2° C. and about 37°C.; in the presence or absence of light, and with or without stirring;e) a fifth step wherein the therapeutic protein is incubated with theactivated water soluble polymer and nucleophilic catalyst underconditions that allow conjugation of the activated water-soluble polymerto one or more oxidized carbohydrates on the therapeutic protein, saidconditions comprising a time period between about 0.5 hours and about 24hours, a temperature between about 2° C. and about 37° C.; in thepresence or absence of light, and with or without stirring; and f) asixth step wherein the conjugating the water soluble polymer to the oneor more oxidized carbohydrates of the therapeutic protein in the fifthstep is stopped by the addition of a quenching agent selected from thegroup consisting of L-cysteine, methionine, glutathione, glycerol,Na₂S₂O₅ (sodium meta bisulfite), tryptophane, tyrosine, histidine orderivatives thereof, kresol, imidazol, and combinations thereof; whereinthe quenching agent is added to result in a final concentration of about1 mM and about 100 mM, under conditions comprising a time period betweenabout 5 minutes and about 120 minutes; a temperature between about 2° C.and about 37° C.; in the presence or absence of light, and with orwithout stirring. In another embodiment, the initial concentration ofthe therapeutic protein in the first step is about 1 mg/ml and the pH isabout 6.0; wherein the final concentration of oxidizing agent in thesecond step is about 400 μM, and the conditions in the fifth stepcomprise a time period of about 10 minutes, a temperature of about 22°C., the absence of light and with stirring; wherein the excessconcentration in the third step is about 50 molar excess; wherein theconditions in the third step comprise a time period of about 15 minutes,a temperature of about 22° C., the absence of light and with stirring;wherein the final concentration of the nucleophilic catalyst in thefourth step is about 10 mM, and the conditions in the fourth stepcomprise a time period of about 15 minutes, a temperature of about 22°C., the absence of light and with stirring; wherein the conditions ofincubating the therapeutic protein with the activated water solublepolymer and nucleophilic catalyst in the fifth step comprise a timeperiod of about 2 hours; a temperature of about 22° C.; the absence oflight; and with stirring; and wherein the quenching agent in the sixthstep is L-cysteine; and wherein the L-cysteine is added to result in afinal concentration of about 10 mM and the conditions in the sixth stepcomprise a time period of about 60 minutes, a temperature of about 22°C., the absence of light and with stirring. In still another embodiment,the water soluble polymer is PSA. In one embodiment, the water solublepolymer is PEG. In another embodiment, the water soluble polymer is HES.In yet another embodiment, the water soluble polymer is HAS. In anotherembodiment the PSA is comprised of about 10-300 sialic acid units. Instill another embodiment, the therapeutic protein is FIX. In anotherembodiment, the therapeutic protein is FVIIa. In yet another embodiment,the therapeutic protein is FVIII.

In another embodiment, an aforementioned method is provided wherein theoxidizing agent is sodium periodate (NaIO₄).

In another embodiment, the aforementioned method is provided wherein theoxidized carbohydrate moiety of the therapeutic protein is located inthe activation peptide of the blood coagulation protein.

In another embodiment, the aforementioned method is provided wherein thePSA comprises an activated aminooxy linker selected from the groupconsisting of: a) a 3-oxa-pentane-1,5-dioxyamine linker of the formula:

b) a 3,6,9-trioxa-undecane-1,11-dioxyamine linker of the formula:

and

c) a 3,6,9,12,15-penatoxa-heptadecane-1,17-dioxyamine linker of theformula:

wherein the PSA is oxidized by incubation with an oxidizing agent toform a terminal aldehyde group at the non-reducing end of the PSA.

In another embodiment, the aminooxy linker is3-oxa-pentane-1,5-dioxyamine.

In another embodiment, the aforementioned method is provided wherein thenucleophilic catalyst is provided at a concentration between about 1 mMand about 50 mM. In another embodiment, the nucleophilic catalyst ism-toluidine. In still another embodiment, the m-toluidine is present inthe conjugation reaction at a concentration of about 10 mM.

In another embodiment, the aforementioned method is provided furthercomprising the step of purifying the conjugated therapeutic protein. Inone embodiment, the conjugated therapeutic protein is purified by amethod selected from the group consisting of chromatography, filtrationand precipitation. In still another embodiment, the chromatography isselected from the group consisting of Hydrophobic InteractionChromatography (HIC), Ion Exchange chromatography (IEC), Size exclusionchromatography (SEC), Affinity chromatography, and Reversed-phasechromatography. In one embodiment, an anti-chaotropic salt is used in achromotography loading step and in a chromatography washing step. Instill another embodiment, the chromatography takes place in a column. Inone embodiment, the column comprises a chromatography resin selectedfrom the group consisting of Phenyl-Sepharose FF and Butyl-Sepharose FF.In yet another embodiment, the resin is present in the column at a bedheight of between about 5 cm and about 20 cm.

In still another embodiment, the bed height is about 10 cm. In anotherembodiment, an aforementioned method is provided wherein comprising oneor more washing steps wherein flow direction is set to up-flow andwherein the flow rate is between about 0.2 cm/min and about 6.7 cm/min.In one embodiment, the flow rate is about 2 cm/min. In anotherembodiment, the aforementioned method is provided comprising one or moreelution steps wherein flow direction is set to down-flow and wherein theflow rate is between about 0.1 cm/min and about 6.7 cm/min. In oneembodiment, the flow rate is about 1 cm/min.

In yet another embodiment, the aforementioned method is provided furthercomprising concentrating the conjugated therapeutic protein byultra-/diafiltration (UF/DF). In another embodiment, the aforementionedmethod is provided wherein the final concentration of therapeuticprotein is between about 0.5 and about 3 mg/ml. In another embodiment,the aforementioned method is provided wherein the therapeutic proteincomprises between about 5 and about 11 water soluble polymer moieties.

In still another embodiment, the aforementioned method is providedwherein the conjugated therapeutic protein is purified usingchromatography; wherein an anti-chaotropic salt is used for a loadingstep and for a washing step; the method comprising one or more washingsteps wherein flow direction is set to up-flow and wherein the flow rateis between about 0.2 cm/min and about 6.7 cm/min and one or more elutionsteps wherein flow direction is set to down-flow and wherein the flowrate is between about 0.2 cm/min and about 6.7 cm/min; furthercomprising concentrating the conjugated therapeutic protein byultra-/diafiltration (UF/DF).

In another embodiment, the aforementioned method is provided wherein thechromatography is hydrophobic interaction chromatography (HIC); whereinthe one or more washing steps flow rate is about 2 cm/min; and whereinthe one or more elution steps flow rate is about 1 cm/min.

A modified therapeutic protein produced by an aforementioned method isalso provided by the present disclosure.

In one embodiment of the present disclosure, a method of preparing awater-soluble polymer containing an active aminooxy group is providedcomprising: a) incubating a solution comprising an oxidizedwater-soluble polymer with an aminooxy linker comprising an activeaminooxy group under conditions that allow the formation of a stableoxime linkage between the oxidized water-soluble polymer and theactivated aminooxy linker, said conditions comprising a time periodbetween about 1 minute and about 24 hours; a temperature between about2° C. and about 8° C.; in the presence or absence of light, and with orwithout stirring; thereby forming a water-soluble polymer containing anactive aminooxy group; and b) purifying the water soluble polymercontaining an active aminooxy group by a method selected from the groupconsisting of chromatography, filtration, dialysis, and precipitation,at a temperature between about 2° C. and about 8° C.; said water solublepolymer is selected from the group consisting of polyethylene glycol(PEG), branched PEG, PolyPEG® (Warwick Effect Polymers; Coventry, UK),polysialic acid (PSA), starch, hydroxyalkyl starch (HAS), hydroxylethylstarch (HES), carbohydrate, polysaccharides, pullulane, chitosan,hyaluronic acid, chondroitin sulfate, dermatan sulfate, starch, dextran,carboxymethyl-dextran, polyalkylene oxide (PAO), polyalkylene glycol(PAG), polypropylene glycol (PPG), polyoxazoline,polyacryloylmorpholine, polyvinyl alcohol (PVA), polycarboxylate,polyvinylpyrrolidone, polyphosphazene, polyoxazoline,polyethylene-co-maleic acid anhydride, polystyrene-co-maleic acidanhydride, poly(1-hydroxymethylethylene hydroxymethylformal) (PHF),2-methacryloyloxy-2′-ethyltrimethylammoniumphosphate (MPC); therebyforming an oxime linkage between the water-soluble polymer and theaminooxy linker.

In another embodiment, the aforementioned method is provided wherein theoxime linkage formation is catalyzed by a nucleophilic catalyst selectedfrom the group consisting of o-amino benzoic acid, m-amino benzoic acid,p-amino benzoic acid, sulfanilic acid, o-aminobenzamide, o-toluidine,m-toluidine, p-toluidine, o-anisidine, m-anisidine, and p-anisidine.

In another embodiment, the aforementioned method is provided wherein thewater soluble polymer is PSA In another embodiment, the aforementionedmethod is provided wherein the water-soluble polymer is PEG. In anotherembodiment, the aforementioned method is provided wherein the solutioncomprising the oxidized water-soluble polymer and the aminooxy linkercomprising an active aminooxy group is incubated at 4° C. for 1 h in theabsence of light with stirring. In another embodiment, theaforementioned method is provided wherein the water soluble polymercontaining an active aminooxy group is purified by anion exchangechromatography at a temperature of 4° C.

In yet another embodiment of the present disclosure, a method ofpreparing a water soluble polymer containing an active aminooxy group isprovided comprising: a) incubating a solution comprising a water-solublepolymer with an aminooxy linker comprising an active aminooxy groupunder conditions that allow the formation of a stable oxime linkagebetween the water-soluble polymer and the activated aminooxy linker,said conditions comprising a time period between about 1 minute andabout 24 hours; a temperature between about 22° C. and about 37° C.; inthe presence or absence of light, and with or without stirring; therebyforming a water-soluble polymer containing an active aminooxy group; andb) purifying the water soluble polymer containing an active aminooxygroup by a method selected from the group consisting of chromatography,filtration, dialysis, and precipitation; thereby forming an oximelinkage between the water-soluble polymer and aminooxy linker.

In another embodiment, the aforementioned method is provided whereinsaid oxime linkage formation is catalyzed by a nucleophilic catalystselected from the group consisting of o-amino benzoic acid, m-aminobenzoic acid, p-amino benzoic acid, sulfanilic acid, o-aminobenzamide,o-toluidine, m-toluidine, p-toluidine, o-anisidine, m-anisidine, andp-anisidine.

In another embodiment, the aforementioned method is provided wherein thesolution comprising the water-soluble polymer and the aminooxy linkercomprising an active aminooxy group is incubated at 22° C. for 2 h inthe absence of light with stirring. In another embodiment, theaforementioned method is provided wherein the solution comprising thewater-soluble polymer and the aminooxy linker comprising an activeaminooxy group is incubated at 22° C. for 2 h in the absence of lightwith stirring; said method further comprising the step of increasing thetemperature of the solution to a temperature between about 32° C. andabout 37° C. and incubating for an additional 12-24 hours. In anotherembodiment, the aforementioned method is provided comprising theadditional step of adding an additional amount of aminooxy linkercomprising an active aminooxy group immediately prior to increasing thetemperature.

In another embodiment, the aforementioned method is provided wherein thewater soluble polymer containing an active aminooxy group is purified bya method selected from the group consisting of dialysis,ultrafiltration/diafiltration (UF/DF), and chromatography at atemperature of 22° C. In another embodiment, the aforementioned methodis provided further comprising the step of purifying the water solublepolymer containing an active aminooxy group by a method selected fromthe group consisting of dialysis, UF/DF or chromatography at 4° C.

In still another embodiment of the present disclosure, a method ofpreparing a PSA-aminooxy reagent aminooxy group is provided comprising:a) incubating a solution comprising an oxidized PSA with an aminooxylinker comprising an active aminooxy group under conditions that allowthe formation of a stable oxime linkage between the oxidized PSA and theactivated aminooxy linker, said conditions comprising a time period 1hour; a temperature of 4° C.; in the absence of light, and withstirring; thereby forming a PSA containing an active aminooxy group; andb) purifying the PSA containing an active aminooxy group by anionexchange chromatography, a temperature of 4° C.; said activated aminooxylinker is 3-oxa-pentane-1,5-dioxyamine linker of the formula:

thereby forming an oxime linkage between the PSA and the aminooxylinker.

In yet another embodiment of the present disclosure, a method ofpreparing a PSA-aminooxy reagent aminooxy group is provided comprising:a) incubating a solution comprising a non-oxidized PSA with an aminooxylinker comprising an active aminooxy group under conditions that allowthe formation of a stable oxime linkage between the non-oxidized PSA andthe activated aminooxy linker, said conditions comprising a time period2 hours; a temperature of 22° C.; in the absence of light, and withstirring; thereby forming a PSA containing an active aminooxy group; andb) purifying the PSA containing an active aminooxy group by dialysis ata temperature of 22° C.; said activated aminooxy linker is3-oxa-pentane-1,5-dioxyamine linker of the formula:

thereby forming an oxime linkage between the non-oxidized PSA and theaminooxy linker.

In still another embodiment of the present disclosure, a method ofconjugating a water soluble polymer to an oxidized carbohydrate moietyof a blood coagulation protein is provided comprising contacting theoxidized carbohydrate moiety with an activated water soluble polymerunder conditions that allow conjugation; said blood coagulation proteinselected from the group consisting of Factor IX (FIX), Factor VIII(FVIII), Factor VIIa (FVIIa), Von Willebrand Factor (VWF), Factor FV(FV), Factor X (FX), Factor XI (FXI), Factor XII (FXII), thrombin (FII),protein C, protein S, tPA, PAI-1, tissue factor (TF) and ADAMTS 13protease or a biologically active fragment, derivative or variantthereof; said water soluble polymer containing an active aminooxy groupand is selected from the group consisting of polyethylene glycol (PEG),branched PEG, polysialic acid (PSA), carbohydrate, polysaccharides,pullulane, chitosan, hyaluronic acid, chondroitin sulfate, dermatansulfate, starch, dextran, carboxymethyl-dextran, polyalkylene oxide(PAO), polyalkylene glycol (PAG), polypropylene glycol (PPG),polyoxazoline, polyacryloylmorpholine, polyvinyl alcohol (PVA),polycarboxylate, polyvinylpyrrolidone, polyphosphazene, polyoxazoline,polyethylene-co-maleic acid anhydride, polystyrene-co-maleic acidanhydride, poly(1-hydroxymethylethylene hydroxymethylformal) (PHF),2-methacryloyloxy-2′-ethyltrimethylammoniumphosphate (MPC); and saidcarbohydrate moiety oxidized by incubation with a buffer comprising anoxidizing agent selected from the group consisting of sodium periodate(NaIO₄), lead tetraacetate (Pb(OAc)₄) and potassium perruthenate(KRuO4); wherein an oxime linkage is formed between the oxidizedcarbohydrate moiety and the active aminooxy group on the water solublepolymer.

FIGURES

FIG. 1 shows the primary structure of coagulation Factor IX (SEQ ID NO:1).

FIG. 2 shows the coupling of oxidized rFIX to aminooxy-PSA.

FIG. 3 shows the synthesis of the water soluble di-aminoxy linkers3-oxa-pentane-1,5-dioxyamine and 3,6,9-trioxa-undecane-1,11-dioxyamine.

FIG. 4 shows the preparation of aminooxy-PSA.

FIG. 5 shows the visualization of PSA-FIX conjugates prepared in thepresence of different catalysts by SDS PAGE. a) Comparison of anilinewith m-toluidine using different concentrations; b) Comparison ofaniline with o-aminobenzoic acid, m-aminobenzoic acid, p-aminobenzoicacid, p-aminobenzamide and sulfanilic acid; c) Comparison of aniline andm-toluidine with o-anisidine and m-anisidine.

FIG. 6 shows percent of polysialylation with various nucleophiliccatalysts.

DETAILED DESCRIPTION OF THE INVENTION

The pharmacological and immunological properties of therapeutic proteinscan be improved by chemical modification and conjugation with polymericcompounds such as polyethylene glycol (PEG), branched PEG, polysialicacid (PSA), hydroxyalkyl starch (HAS), hydroxylethyl starch (HES),carbohydrate, polysaccharides, pullulane, chitosan, hyaluronic acid,chondroitin sulfate, dermatan sulfate, starch, dextran,carboxymethyl-dextran, polyalkylene oxide (PAO), polyalkylene glycol(PAG), polypropylene glycol (PPG), polyoxazoline,polyacryloylmorpholine, polyvinyl alcohol (PVA), polycarboxylate,polyvinylpyrrolidone, polyphosphazene, polyoxazoline,polyethylene-co-maleic acid anhydride, polystyrene-co-maleic acidanhydride, poly(1-hydroxymethylethylene hydroxymethylformal) (PHF),2-methacryloyloxy-2′-ethyltrimethylammoniumphosphate (MPC). Theproperties of the resulting conjugates generally strongly depend on thestructure and the size of the polymer. Thus, polymers with a defined andnarrow size distribution are usually preferred in the art. Syntheticpolymers like PEG can be manufactured easily with a narrow sizedistribution, while PSA can be purified in such a manner that results ina final PSA preparation with a narrow size distribution. In additionPEGylation reagents with defined polymer chains and narrow sizedistribution are on the market and commercially available for areasonable price.

The addition of a soluble polymer, such as through polysialylation, isone approach to improve the properties of therapeutic proteins such asthe blood coagulation protein FIX, as well as other coagulation proteins(e.g., VWF, FVIIa (see, e.g., US 2008/0221032A1, incorporated herein byreference) and FVIII).

Therapeutic Proteins

In certain embodiments of the invention, the aforementioned polypeptidesand polynucleotides are exemplified by the following therapeuticproteins: enzymes, antigens, antibodies, receptors, blood coagulationproteins, growth factors, hormones, and ligands. In certain embodiments,the therapeutic protein is a blood coagulation protein such as Factor IX(FIX), Factor VIII (FVIII), Factor VIIa (FVIIa), Von Willebrand Factor(VWF), Factor FV (FV), Factor X (FX), Factor XI (FXI), Factor XII(FXII), thrombin (FII), protein C, protein S, tPA, PAI-1, tissue factor(TF) or ADAMTS 13 protease. In one embodiment, a therapeutic proteinaccording to the invention is a glycoprotein or, in various embodiments,a protein that is not naturally glycosylated in vivo (i.e., a proteinthat does not contain a natural glycosylation site or a protein that isnot glycosylated in a host cell prior to purification).

In certain embodiments, the therapeutic protein is immunoglobulins,cytokines such IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-5, IL-6,IL-11, colony stimulating factor-1 (CSF-1), M-CSF, SCF, GM-CSF,granulocyte colony stimulating factor (G-CSF), EPO, interferon-alpha(IFN-alpha), consensus interferon, IFN-beta, IFN-gamma, IFN-omega, IL-7,IL-8, IL-9, IL-10, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18,IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-31, IL-32 alpha, IL-33,thrombopoietin (TPO), angiopoietins, for example Ang-1, Ang-2, Ang-4,Ang-Y, the human angiopoietin-like polypeptides ANGPTL1 through 7,vitronectin, vascular endothelial growth factor (VEGF), angiogenin,activin A, activin B, activin C, bone morphogenic protein-1, bonemorphogenic protein-2, bone morphogenic protein-3, bone morphogenicprotein-4, bone morphogenic protein-5, bone morphogenic protein-6, bonemorphogenic protein-7, bone morphogenic protein-8, bone morphogenicprotein-9, bone morphogenic protein-10, bone morphogenic protein-11,bone morphogenic protein-12, bone morphogenic protein-13, bonemorphogenic protein-14, bone morphogenic protein-15, bone morphogenicprotein receptor IA, bone morphogenic protein receptor IB, bonemorphogenic protein receptor II, brain derived neurotrophic factor,cardiotrophin-1, ciliary neutrophic factor, ciliary neutrophic factorreceptor, cripto, cryptic, cytokine-induced neutrophil chemotacticfactor 1, cytokine-induced neutrophil, chemotactic factor 2α,cytokine-induced neutrophil chemotactic factor 2β, β endothelial cellgrowth factor, endothelin 1, epidermal growth factor, epigen,epiregulin, epithelial-derived neutrophil attractant, fibroblast growthfactor 4, fibroblast growth factor 5, fibroblast growth factor 6,fibroblast growth factor 7, fibroblast growth factor 8, fibroblastgrowth factor 8b, fibroblast growth factor 8c, fibroblast growth factor9, fibroblast growth factor 10, fibroblast growth factor 11, fibroblastgrowth factor 12, fibroblast growth factor 13, fibroblast growth factor16, fibroblast growth factor 17, fibroblast growth factor 19, fibroblastgrowth factor 20, fibroblast growth factor 21, fibroblast growth factoracidic, fibroblast growth factor basic, glial cell line-derivedneutrophic factor receptor α1, glial cell line-derived neutrophic factorreceptor α2, growth related protein, growth related protein α, growthrelated protein β, growth related protein γ, heparin binding epidermalgrowth factor, hepatocyte growth factor, hepatocyte growth factorreceptor, hepatoma-derived growth factor, insulin-like growth factor I,insulin-like growth factor receptor, insulin-like growth factor II,insulin-like growth factor binding protein, keratinocyte growth factor,leukemia inhibitory factor, leukemia inhibitory factor receptor α, nervegrowth factor nerve growth factor receptor, neuropoietin,neurotrophin-3, neurotrophin-4, oncostatin M (OSM), placenta growthfactor, placenta growth factor 2, platelet-derived endothelial cellgrowth factor, platelet derived growth factor, platelet derived growthfactor A chain, platelet derived growth factor AA, platelet derivedgrowth factor AB, platelet derived growth factor B chain, plateletderived growth factor BB, platelet derived growth factor receptor α,platelet derived growth factor receptor β, pre-B cell growth stimulatingfactor, stem cell factor (SCF), stem cell factor receptor, TNF,including TNF0, TNF1, TNF2, transforming growth factor α, transforminggrowth factor β, transforming growth factor β1, transforming growthfactor β1.2, transforming growth factor β2, transforming growth factorβ3, transforming growth factor β5, latent transforming growth factor β1,transforming growth factor β binding protein I, transforming growthfactor β binding protein II, transforming growth factor β bindingprotein III, thymic stromal lymphopoietin (TSLP), tumor necrosis factorreceptor type I, tumor necrosis factor receptor type II, urokinase-typeplasminogen activator receptor, vascular endothelial growth factor, andchimeric proteins and biologically or immunologically active fragmentsthereof.

In certain embodiments, the therapeutic protein is alpha-, beta-, andgamma-interferons, colony stimulating factors including granulocytecolony stimulating factors, fibroblast growth factors, platelet derivedgrowth factors, phospholipase-activating protein (PUP), insulin, plantproteins such as lectins and ricins, tumor necrosis factors and relatedalleles, soluble forms of tumor necrosis factor receptors, interleukinreceptors and soluble forms of interleukin receptors, growth factorssuch as tissue growth factors, such as TGFαs or TGFβs and epidermalgrowth factors, hormones, somatomedins, pigmentary hormones,hypothalamic releasing factors, antidiuretic hormones, prolactin,chorionic gonadotropin, follicle-stimulating hormone,thyroid-stimulating hormone, tissue plasminogen activator, andimmunoglobulins such as IgG, IgE, IgM, IgA, and IgD, a galactosidase,α-galactosidase, β-galactosidase, DNAse, fetuin, leutinizing hormone,estrogen, corticosteroids, insulin, albumin, lipoproteins, fetoprotein,transferrin, thrombopoietin, urokinase, DNase, integrins, thrombin,hematopoietic growth actors, leptin, glycosidases, Humira (adalimumab),Prolia (denosumab), Enbrel (etanercept), and fragments thereof, or anyfusion proteins comprising any of the above mentioned proteins orfragments thereof. In addition to the aforementioned proteins, thefollowing Table 1 provides therapeutic proteins contemplated by thepresent invention:

TABLE 1 Follicular dendritic cell secreted peptideAngiotensin-converting enzyme Interleukin-1 family member 6 HerstatinDermokine Antithrombin-III Prostate and testis expressed protein 2Leucine-rich repeat-containing protein 28 Secreted frizzled-relatedprotein 1 Apolipoprotein B-100 Group XIIA secretory phospholipase A2LRRN4 C-terminal-like protein Ectodysplasin-A Apolipoprotein D Collagenalpha-3(V) chain Ly6/PLAUR domain-containing protein 2 Secretedfrizzled-related protein 2 Apolipoprotein E Alpha-2-macroglobulin-likeprotein 1 Transmembrane protein 81 ResistinBeta-1,4-galactosyltransferase 1 Dermatopontin Myelin protein zero-likeprotein 3 Osteopontin Bone morphogenetic protein 7 Cartilage-associatedprotein Protein notum homolog Secreted frizzled-related protein 5Complement C1q subcomponent subunit B Desert hedgehog proteinUDP-glucuronosyltransferase 3A2 Secreted frizzled-related protein 4C4b-binding protein alpha chain Extracellular matrix protein 2Protocadherin alpha-1 Secreted phosphoprotein 24 Calreticulin Gastricintrinsic factor Phospholipase D4 Glypican-6 Corticosteroid-bindingglobulin Interleukin-33 Retinol dehydrogenase 10 Secretedfrizzled-related protein 3 Carboxypeptidase A1 Bone morphogeneticprotein 2 Sialic acid-binding Ig-like lectin 14 C-C motif chemokine 4Carboxypeptidase A2 Bone morphogenetic protein 6 Transmembrane protein161A Melanocyte protein Pmel 17 Eotaxin Uncharacterized protein KIAA0564Transmembrane protein 161B Secreted Ly-6/uPAR-related protein 1 C-Cmotif chemokine 13 Cerberus Transmembrane protein 182Beta-microseminoprotein C-C motif chemokine 18 Carbohydratesulfotransferase 8 Protein FAM24B Glypican-4 C-C motif chemokine 20Contactin-associated protein-like 3 Transmembrane protein 52 Tumornecrosis factor ligand superfamily Triggering receptor expressed onGroup XIIB secretory phospholipase A2-like protein Major facilitatorsuperfamily domain- member 15 myeloid cells 2 containing protein 4Resistin-like beta C-C motif chemokine 2 CorticoliberinUDP-glucuronosyltransferase 2A3 Tumor necrosis factor ligand superfamilyTransforming growth factor-beta-induced A disintegrin andmetalloproteinase with Odontogenic ameloblast-associated member 12protein ig-h3 thrombospondin motifs 19 protein SPARC CD40 ligand UPF0556protein C19orf10 Neurosecretory protein VGF Glypican-5 CorneodesmosinC—X—C motif chemokine 3 Secreted phosphoprotein 2, 24 kDa Anteriorgradient protein 2 homolog Complement factor D Cystatin-M ProteinFAM150B Protein canopy homolog 2 Chromogranin-A Defensin-5Growth/differentiation factor 9 Glypican-1 Collagen alpha-1(I) chainDefensin-6 Clusterin-like protein 1 von Willebrand factor Adomain-containing Disintegrin and metalloproteinase domain- Adisintegrin and metalloproteinase with Transmembrane and immunoglobulinprotein 2 containing protein 18 thrombospondin motifs 18domain-containing protein 2 WNT1-inducible-signaling pathwayCysteine-rich secretory protein LCCL A disintegrin and metalloproteinasewith C-type lectin domain-containing protein protein 1 domain-containing1 thrombospondin motifs 3 UNQ5810/PRO19627 C-C motif chemokine 1Collagen alpha-4(IV) chain Dickkopf-related protein 4Epididymal-specific lipocalin-10 SPARC-related modular calcium-bindingKeratinocyte differentiation-associated protein A disintegrin andmetalloproteinase with thrombospondin motifs 5 A disintegrin andmetalloproteinase with thrombospondin protein 2 motifs 8 C-type lectindomain family 11 member A Complement C4-B Mammalian ependymin-relatedprotein 1 Epididymal-specific lipocalin-8 Secreted Ly-6/uPAR-relatedprotein 2 Collagen alpha-2(V) chain Fibrillin-3 Basic proline-richpeptide P-E Glypican-3 Complement C5 Fetuin-B Putative uncharacterizedprotein C10orf99 Secreted and transmembrane protein 1 Collagenalpha-1(VII) chain Fibroblast growth factor 6 Uncharacterized proteinC17orf77 Testis-expressed sequence 264 protein Complement component C7Keratinocyte growth factor Arylacetamide deacetylase-like 2 Glypican-2Complement component C8 beta chain Growth/differentiation factor 8Epididymal-specific lipocalin-12 Serine protease 23 Complement componentC8 gamma chain Gastric inhibitory polypeptide B melanoma antigen 2 39Sribosomal protein L55, mitochondrial Collagen alpha-1(XV) chainGlycoprotein hormone beta-5 B melanoma antigen 3 Protein NipSnap homolog3A Collagen alpha-1(XVI) chain Granzyme M Bovine seminal plasma proteinhomolog 1 Fibronectin Collagen alpha-1(XVIII) chain Gastrin-releasingpeptide Complement C1q-like protein 3 Neudesin Collagen alpha-1(XIX)chain Serine protease HTRA1 UPF0565 protein C2orf69 Fibroblast growthfactor receptor 2 Cartilage oligomeric matrix protein Interferon alpha-4UPF0669 protein C6orf120 Carbonic anhydrase 6 C-reactive proteinInterferon alpha-5 Colipase-like protein C6orf127 Deleted in malignantbrain tumors 1 protein Granulocyte colony-stimulating factor Interferonalpha-7 Uncharacterized protein C7orf69 SPARC-related modularcalcium-binding Granulocyte-macrophage colony- A disintegrin andmetalloproteinase with Platelet-derived growth factor receptor-likeprotein 1 stimulating factor thrombospondin motifs 7 protein Amyloidbeta A4 protein Protein CYR61 Immunoglobulin superfamily member 10Chondroadherin-like protein Tumor necrosis factor receptor superfamilyComplement component receptor 1-like Protease-associateddomain-containing Putative uncharacterized protein member 6 proteinprotein of 21 kDa UNQ6490/PRO21339 Gamma-aminobutyric acid type Breceptor Stem cell growth factor; lymphocyte Abhydrolasedomain-containing protein Putative uncharacterized protein subunit 1secreted C-type lectin FAM108A1 UNQ6493/PRO21345 Pro-neuregulin-1,membrane-bound CMP-N-acetylneuraminate-beta- A disintegrin andmetalloproteinase with Putative uncharacterized protein isoformgalactosamide-alpha-2,3-sialyltransferase thrombospondin motifs 9UNQ5815/PRO19632 Glycoprotein hormone alpha-2 Dipeptidyl peptidase 4Interleukin-9 receptor Cystatin-A Membrane metallo-endopeptidase-like 1Dentin sialophosphoprotein Interleukin-9 Peptidase inhibitor R3HDML Fcreceptor-like A Endothelin-1 Inhibin beta B chain Cystatin-9 C-C motifchemokine 4-like Ephrin-B1 Serine protease inhibitor Kazal-type 2 DANdomain family member 5 Epithelial discoidin domain-containingEpidermis-specific serine protease-like BMP-binding endothelialregulator Insulin-like growth factor-binding protein- receptor 1 proteinprotein like 1 Mucin-1 EMILIN-1 Keratinocyte-associated protein 2Epididymal sperm-binding protein 1 Vascular endothelial growth factor AEndoplasmin Laminin subunit alpha-1 Elafin Fibulin-1 Ephrin type-Areceptor 3 Leukocyte cell-derived chemotaxin-2 Protein FAM55A Prolactinreceptor Ephrin type-B receptor 6 Gastric triacylglycerol lipaseGrowth/differentiation factor 6 Proprotein convertase subtilisin/kexinGlycosyltransferase 1 domain-containing Leucine-rich repeat and calponinGlucose-fructose oxidoreductase domain- type 6 protein 1 homologydomain-containing protein 3 containing protein 1 CD209 antigenCoagulation factor X Pancreatic lipase-related protein 2 ErythropoietinCollagen alpha-2(XI) chain Coagulation factor VIII Epididymis-specificalpha-mannosidase Glutathione peroxidase 6 Granulocyte-macrophagecolony- Complement C1q tumor necrosis factor- Fibronectin type IIIdomain-containing Uncharacterized protein stimulating factor receptorsubunit alpha related protein 7 protein 7 UNQ511/PRO1026 ElastinFibrillin-2 Microfibrillar-associated protein 5 Beta-defensin 128Interleukin-15 receptor subunit alpha Alpha-2-HS-glycoproteinMuellerian-inhibiting factor Interleukin-31 Midkine Fibroblast growthfactor 10 Matrix metalloproteinase-21 Interleukin-34 Integrin alpha-7Fibrinogen alpha chain Matrix metalloproteinase-17 Plasmakallikrein-like protein 4 Mucin-4 Fibrinogen beta chain Matrixmetalloproteinase-20 Epididymal-specific lipocalin-9 Peptidyl-glycinealpha-amidating Long palate, lung and nasal epitheliumN-acetylglucosamine-1- cDNA FLJ60957, highly similar to monooxygenasecarcinoma-associated protein 1 phosphotransferase subunit gamma Secretedfrizzled-related protein 4 Apolipoprotein A-I Gastrin Multimerin-2Lipase member M Proteoglycan 4 Glycoprotein hormones alpha chainPromotilin CLECSF12 Tumor necrosis factor receptor superfamilyN-acetylglucosamine-1- FRAS1-related extracellular matrix Putativeinactive group IIC secretory member 25 phosphotransferase subunitsalpha/beta protein 3 phospholipase A2 Attractin Granzyme A Proteinkinase C-binding protein NELL1 Serine protease MPN2 Prostate-associatedmicroseminoprotein Hepatocyte growth factor-like protein Protein kinaseC-binding protein NELL2 Netrin-5 Alpha-amylase 1 Insulin-like growthfactor-binding protein 1 Neurotrypsin NHL repeat-containing protein 3Brain-derived neurotrophic factor Insulin-like growth factor-bindingprotein 2 Neuroserpin Olfactomedin-like protein 2B C-type lectin domainfamily 4 member M Insulin-like growth factor-binding protein 4 Nidogen-2Ovochymase-2 Granulocyte colony-stimulating factor Tumor necrosis factorreceptor superfamily Abhydrolase domain-containing protein Putativeuncharacterized protein receptor member 10D FAM108B1 UNQ3029/PRO9830Insulin-like growth factor II Interferon alpha-1/13 Neurotrophin-4Ovochymase-1 Carcinoembryonic antigen-related cell Interferon-inducedhelicase C domain- Epididymal secretory glutathione Putativepregnancy-specific beta-1- adhesion molecule 1 containing protein 1peroxidase glycoprotein 7 C-type lectin domain family 7 member AInterferon alpha-2 Group 10 secretory phospholipase A2 Ovostatin homolog2 CMRF35-like molecule 1 Interferon beta Group IID secretoryphospholipase A2 Orexigenic neuropeptide QRFP Choline transporter-likeprotein 4 Interferon gamma Lactoperoxidase Lymphocyte antigen 6KPulmonary surfactant-associated protein A1 Insulin-like growth factor IBp53 apoptosis effector related to PMP-22 Prostate and testis expressedprotein 1 Spermine oxidase Indian hedgehog protein Placenta-specificprotein 1 Putative phospholipase B-like 1CMP-N-acetylneuraminate-beta-1,4- Neural cell adhesion moleculeTuberoinfundibular peptide of Putative uncharacterized proteingalactoside alpha-2,3-sialyltransferase L1-like protein 39 residuesFLJ42147 Kallikrein-8 Interleukin-13 Prolargin Otogelin Tissue-typeplasminogen activator Interleukin-2 Secretogranin-2 Ribonuclease 8Peroxisomal N(1)-acetyl- Chymotrypsin-like elastase family Endonucleasedomain-containing 1 Nuclear pore complex-interacting protein-spermine/spermidine oxidase member 2A protein like 2 Probablepalmitoyltransferase ZDHHC4 Inhibin beta A chain Semaphorin-3BProactivator polypeptide-like 1 Cholesteryl ester transfer proteinPancreatic secretory trypsin inhibitor Somatostatin Protein spinsterhomolog 2 HLA class I histocompatibility antigen, A-2 Tumor necrosisfactor receptor superfamily Dehydrogenase/reductase SDR family vonWillebrand factor C domain- alpha chain member 21 member 4-like 2containing protein 2-like Collagen alpha-1(II) chain Inter-alpha-trypsininhibitor heavy chain H1 Transcobalamin-1 Urotensin-2BPro-interleukin-16 Inter-alpha-trypsin inhibitor heavy chain H2 Trefoilfactor 2 Tetraspanin-18 Leptin receptor Inter-alpha-trypsin inhibitorheavy chain H3 Testican-1 UPF0514 membrane protein FAM159A DecorinProstate-specific antigen Serum paraoxonase/lactonase 3 Latherin Stromalcell-derived factor 1 Kallikrein-4 Tolloid-like protein 2Methyltransferase-like protein 7B Tenascin Plasma kallikrein Trypsin-2Protein TEX261 Disintegrin and metalloproteinase domain-Calcium-activated chloride channel RING finger and SPRY domain-Alkylated DNA repair protein alkB containing protein 12 regulator 4containing protein 1 homolog 7 A disintegrin and metalloproteinase withBactericidal/permeability-increasing Calcium-binding and coiled-coildomain- Transmembrane emp24 domain- thrombospondin motifs 13protein-like 1 containing protein 1 containing protein 6 T-cell surfaceglycoprotein CD8 alpha chain Leptin Protein Wnt-2 XK-related protein 5EGFR-coamplified and overexpressed A disintegrin and metalloproteinasewith Ectonucleoside triphosphate Putative V-set and immunoglobulinprotein thrombospondin motifs 4 diphosphohydrolase 8 domain-containingprotein 7 Autophagy-related protein 16-1 Hepatic triacylglycerol lipaseProtein Wnt-8b Insulin growth factor-like family member 3 Breast canceranti-estrogen resistance Lymphocyte antigen 6 complex locusUDP-GlcNAc:betaGal beta-1,3-N- Nuclear pore complex-interacting protein-protein 3 protein G6c acetylglucosaminyltransferase 4 like 1 Cadherin-23Eosinophil lysophospholipase EMI domain-containing protein 1 Secretedphosphoprotein 1 Macrophage colony-stimulating factor 1 Lutropin subunitbeta Uncharacterized protein C6orf15 Collagen alpha-5(VI) chain Folatereceptor alpha Microfibrillar-associated protein 1 Collectin-10 Bmelanoma antigen 5 Low-density lipoprotein receptor-relatedMesencephalic astrocyte-derived Long-chain-fatty-acid--CoA ligase WAPfour-disulfide core domain protein protein 8 neurotrophic factor ACSBG210A E3 ubiquitin-protein ligase LRSAM1 Matrix Gla proteinOncoprotein-induced transcript 3 protein UPF0369 protein C6orf57 Neuralcell adhesion molecule 1 72 kDa type IV collagenase Peptidase inhibitor15 Putative uncharacterized protein C10orf31 Neuroligin-4, X-linkedStromelysin-1 Proline-rich acidic protein 1 Putative uncharacterizedprotein C11orf45 Netrin-G1 Neutrophil collagenase UrocortinUncharacterized protein C12orf28 GPI transamidase component PIG-TMesothelin Trypsin-X3 (EC 3.4.21.4) Uncharacterized protein C17orf67 Kitligand Mucin-5AC HHIP-like protein 2 Beta-defensin 121 Seizure 6-likeprotein Mucin-6 Fractalkine Beta-defensin 130 SLAM family member 7Norrin Protein Wnt-11 Histidine triad nucleotide-binding protein 2 Tumornecrosis factor Oxytocin-neurophysin 1 Protein Wnt-7a Apelin UromodulinBeta-nerve growth factor FCH and double SH3 domains protein 1Placenta-specific protein 9 Tumor necrosis factor ligand superfamilyTumor necrosis factor ligand superfamily Hepatoma-derived growthfactor-related Hepatocellular carcinoma-associated member 13 member 18protein 2 protein TD26 Protein CREG1 Neurotrophin-3 Interleukin-12subunit alpha Persephin EGF-like domain-containing protein 8Platelet-derived growth factor subunit A UPF0577 protein KIAA1324Regulated endocrine-specific protein 18 Aminoacyl tRNA synthetasecomplex- Phosphopantothenoylcysteine Complement C1q tumor necrosisfactor- Complement C1q tumor necrosis factor- interactingmultifunctional protein 1 decarboxylase related protein 9 relatedprotein 8 ADAMTS-like protein 4 Plasminogen activator inhibitor 1Mucin-17 Bone morphogenetic protein 8A Coagulation factor XI Plasminogenactivator inhibitor 2 Lysosomal protein NCU-G1 Protein WFDC13Interleukin-22 receptor subunit alpha-2 Procollagen C-endopeptidaseenhancer 1 Prolyl 4-hydroxylase subunit alpha-3 Protein Wnt-8a Deformedepidermal autoregulatory factor 1 Transmembrane and ubiquitin-likedomain- Peptidyl-prolyl cis-trans isomerase Ig-like domain-containingprotein homolog containing protein 2 SDCCAG10 ENSP00000270642Prostaglandin-H2 D-isomerase Protein disulfide-isomerase Peptidaseinhibitor 16 Abhydrolase domain-containing protein 15Alpha-1-antitrypsin Pigment epithelium-derived factor Poliovirusreceptor-related protein 4 Ribonuclease-like protein 9Alpha-1-antichymotrypsin Pepsin A Solute carrier family 22 member 15Uncharacterized protein C2orf66 Acyl-CoA-binding protein Gastricsin GPIinositol-deacylase Uncharacterized protein C17orf99 Complement factor BSonic hedgehog protein Transmembrane protein 43 Protein FAM150AChoriogonadotropin subunit beta Peptidoglycan recognition proteinI-alpha Angiopoietin-related protein 2 Placenta-specific 1-like proteinVersican core protein Biglycan Angiopoietin-related protein 6Uncharacterized protein C18orf20 Epidermal growth factor receptorProlactin-inducible protein Arylsulfatase K Beta-defensin 110 Ecto-NOXdisulfide-thiol exchanger 2 Platelet factor 4 Augurin Neuritin-likeprotein Hyaluronidase-1 Plasminogen Brain-specific serine protease 4Histidine-rich carboxyl terminus protein 1 Interleukin-1 receptorantagonist protein Serum paraoxonase/arylesterase 1 DBH-likemonooxygenase protein 1 C-type lectin domain family 2 member AInterleukin-6 receptor subunit beta Alkaline phosphatase, placental typeUncharacterized protein C1orf56 Leucine-rich repeat-containing protein70 Interleukin-1 receptor-like 1 Peptidyl-prolyl cis-trans isomerase BCerebellin-3 Serpin A13 Insulin Bone marrow proteoglycan Cerebellin-4BTB/POZ domain-containing protein 17 Glycodelin Basic salivaryproline-rich protein 1 Colipase-like protein C6orf126 Uncharacterizedprotein C12orf53 Parathyroid hormone-related protein Pulmonarysurfactant-associated protein C Uncharacterized protein C11orf83 C-typelectin domain family 9 member A Nurim Parathyroid hormoneUncharacterized protein C16orf89 Complement C1q-like protein 4 Prolyl4-hydroxylase subunit alpha-2 Serum amyloid P-componentCarboxypeptidase-like protein X2 CMRF35-like molecule 4 CD276 antigenSecretogranin-1 Cystatin-9-like Protein FAM151B Cysteine-rich withEGF-like domain Basement membrane-specific heparanDehydrogenase/reductase SDR family Abhydrolase domain-containing proteinprotein 1 sulfate proteoglycan core protein member 13 FAM108A2/A3 CUBand sushi domain-containing protein 1 Antileukoproteinase Beta-defensin123 Osteocrin Ficolin-2 Stabilin-1 Beta-defensin 132 Transmembraneprotease, serine 11E2 Fc receptor-like protein 5 Extracellularsuperoxide dismutase [Cu—Zn] Cytokine-like protein 1 Transmembraneprotein 14E Protein GPR89 Somatotropin Dickkopf-related protein 2Transmembrane protein 207 Junctional adhesion molecule A Serpin B5Dickkopf-like protein 1 TOMM20-like protein 1 Leucine-richrepeat-containing protein 8A Spondin-1 Epididymal secretory proteinE3-beta Uncharacterized protein C3orf41 Multiple inositol polyphosphateStructural maintenance of chromosomes EGF-like repeat and discoidinI-like Submaxillary gland androgen-regulated phosphatase 1 protein 3domain-containing protein 3 protein 3A Neuropilin-1 Syntaxin-1A ProteinFAM55D B melanoma antigen 1 Plexin-A4 Tetranectin Fibroblast growthfactor 17 Inactive carboxylesterase 4 Plexin-B1 Transforming growthfactor beta-1 Fibroblast growth factor 22 Four-jointed box protein 1Periostin Thyroglobulin Fibroblast growth factor-binding protein 2Protein HSN2 Protein RIC-3 Metalloproteinase inhibitor 1Growth/differentiation factor 3 Humanin SLIT and NTRK-like protein 2Metalloproteinase inhibitor 2 GLIPR1-like protein 1 Kielin/chordin-likeprotein Sulfatase-modifying factor 1 Metalloproteinase inhibitor 3Serine protease inhibitor Kazal-type 6 UPF0624 protein C6orf186Sulfatase-modifying factor 2 Urokinase-type plasminogen activatorInterleukin-17B Putative neurofibromin 1-like protein 4/6 Transmembraneprotease, serine 6 Lactotransferrin Interleukin-17C Peroxidasin-likeprotein Lymphotoxin-alpha Trypsin-1 Interleukin-17D SCO-spondin Tumornecrosis factor receptor superfamily Submaxillary glandandrogen-regulated Hyaluronan and proteoglycan link Putativeuncharacterized protein member 10B protein 3B protein 3 UNQ9165/PRO28630Urokinase plasminogen activator surface Tumor necrosis factor receptorsuperfamily Vitelline membrane outer layer protein 1 Calcium-activatedchloride channel receptor member 1A homolog regulator family member 3V-set domain-containing T-cell activation Vascular endothelial growthfactor Choriogonadotropin subunit beta Probable serine proteaseinhibitor 1 receptor 1 variant 1 UNQ9391/PRO34284 Glucagon VitaminD-binding protein Lysozyme-like protein 1 Uncharacterized proteinC4orf26 N-acetylmuramoyl-L-alanine amidase Vitronectin Matrixmetalloproteinase-28 Uncharacterized protein C4orf40 Sulfhydryl oxidase1 von Willebrand factor Nephronectin Uncharacterized protein C5orf55Dehydrogenase/reductase SDR family Lymphocyte antigen 6 complex locusWAP four-disulfide core domain Putative macrophage-stimulating proteinmember 4 protein G5c protein 12 MSTP9 Interleukin-18-binding proteinZinc-alpha-2-glycoprotein Olfactomedin-like protein 1 Uncharacterizedprotein C15orf61 Kin of IRRE-like protein 2 Uncharacterized proteinC14orf93 Olfactomedin-like protein 2A Chymotrypsinogen B2Myeloid-associated differentiation marker Retinoschisin Serine protease27 Beta-defensin 108A Chordin Alpha-1,3-mannosyltransferase ALG2Secretoglobin family 3A member 2 Beta-defensin 1111-acyl-sn-glycerol-3-phosphate C-type lectin domain family 11, member A,A disintegrin and metalloproteinase with Putative V-set andimmunoglobulin acyltransferase gamma isoform CRA_b thrombospondin motifs2 domain-containing protein 6 Advanced glycosylation end product- Majorfacilitator superfamily domain- Disintegrin and metalloproteinase Serineprotease inhibitor Kazal-type specific receptor containing protein 7domain-containing protein 28 5-like 3 NLR family CARD domain-containingLeucine-rich repeat transmembrane Bactericidal/permeability-increasingPutative serine protease inhibitor Kazal- protein 4 neuronal protein 1protein-like 2 type 5-like 2 Pro-neuregulin-2, membrane-bound NADHdehydrogenase [ubiquinone] 1 beta Acid sphingomyelinase-likeDehydrogenase/reductase SDR family isoform subcomplex subunit 11,mitochondrial phosphodiesterase 3b member 7C Sperm-associated antigen11A UPF0546 membrane protein C1orf91 Serine protease inhibitorKazal-type 7 Beta-defensin 131 Oocyte-secreted protein 1 homologCarbonic anhydrase-related protein 10 Neurexophilin-4 Beta-defensin 134Serum albumin Cholecystokinin Protein Wnt-9b Beta-defensin 136 CochlinCodanin-1 Zymogen granule protein 16 homolog B Beta-defensin 116 Plasmaprotease C1 inhibitor Uncharacterized protein C6orf89 Semaphorin-3DProtein FAM132A Interleukin-7 receptor subunit alpha Chondroitin sulfateglucuronyltransferase Apolipoprotein L4 Protein FAM132BInter-alpha-trypsin inhibitor heavy chain H5 Chitinase domain-containingprotein 1 Transmembrane protease, serine 11D Beta-defensin 115Platelet-derived growth factor D Transmembrane protein C9orf7Scrapie-responsive protein 1 Beta-defensin 114 Protein S100-A7CMRF35-like molecule 9 Putative annexin A2-like protein Serine proteaseinhibitor Kazal-type 9 Sialic acid-binding Ig-like lectin 10 CytochromeP450 2S1 Bone morphogenetic protein 10 Lipase member NTubulointerstitial nephritis antigen-like Crumbs protein homolog 3Secretogranin-3 Pancreatic lipase-related protein 3 Tumor necrosisfactor ligand superfamily Dehydrogenase/reductase SDR family ComplementC1q tumor necrosis factor- Testis, prostate and placenta-expressedmember 13B member 7 related protein 4 protein Long-chain-fatty-acid--CoAligase 5 Protein ENED Uncharacterized protein C1orf54 Neuromedin-SClaudin-14 Complement factor H-related protein 4 Carboxypeptidase A6Neuropeptide S Leucine-rich repeat-containing protein 20 Leucine-richrepeat LGI family member 3 C-C motif chemokine 19 Neuronalpentraxin-like protein C16orf38 Interleukin-1 family member 7 GliomedinC-C motif chemokine 25 Otolin-1 Lymphocyte antigen 6 complex locusGlycerophosphodiester phosphodiesterase Chymotrypsin-like elastasefamily Iron/zinc purple acid phosphatase-like protein G5bdomain-containing protein 5 member 2B protein AcetylcholinesteraseProbable G-protein coupled receptor 113 Protein CEI Ovostatin homolog 1Amelogenin, X isoform Probable G-protein coupled receptor 114Uncharacterized protein C6orf1 Plasminogen-related protein A AngiogeninGlycerol-3-phosphate acyltransferase 4 Uncharacterized protein C7orf34Polyserase-3 Anthrax toxin receptor 2 Gremlin-1 Keratinocyte-associatedprotein 3 Putative peptide YY-2 Annexin A2 Potassium channel subfamily Kmember 17 Uncharacterized protein C9orf47 Putative peptide YY-3Apolipoprotein C-III KDEL motif-containing protein 2 Collagenalpha-1(VIII) chain Ribonuclease-like protein 10 Apolipoprotein L1Layilin Uncharacterized protein C18orf54 Ribonuclease-like protein 12Complement C1q subcomponent subunit A Leucine-rich repeat-containingprotein 8B Cystatin-like 1 Ribonuclease-like protein 13 Complement C1qsubcomponent subunit C Leucine-rich repeat-containing protein 8D C2domain-containing protein 2 Serpin A11 Calcitonin Sialic acid-bindingIg-like lectin 6 DDRGK domain-containing protein 1 Kunitz-type proteaseinhibitor 4 Soluble calcium-activated nucleotidase 1 Pregnancy-specificbeta-1-glycoprotein 2 Protein FAM55C Meteorin-like protein C-C motifchemokine 15 Ly6/PLAUR domain-containing protein 1 Collagenalpha-1(XXVI) chain Putative testis serine protease 2 CD97 antigen (Ly6/PLAUR domain-containing protein 5 Protein FAM19A2 Beta-defensin 112Contactin-4 MLN64 N-terminal domain homolog Protein FAM5BUncharacterized protein FLJ37543 Complement C2 Macrophage migrationinhibitory factor Fibroblast growth factor 5 Protein FAM24A Collagenalpha-6(IV) chain 2-acylglycerol O-acyltransferase 3 Probable serineprotease HTRA3 Secreted frizzled-related protein 4 Collagen alpha-2(VI)chain Mitochondrial carrier homolog 1 Interleukin-1 family member 8Complement C1q-like protein 2 Collagen alpha-1(XI) chain ApolipoproteinL6 Serine protease inhibitor Kazal-type 4 Putative uncharacterizedprotein C17orf69 Crumbs homolog 1 Protocadherin alpha-6 OtospiralinPutative cystatin-13 Cystatin-C Protocadherin gamma-A12 Liver-expressedantimicrobial peptide 2 Beta-defensin 109 Neutrophil defensin 1Voltage-gated hydrogen channel 1 Lysyl oxidase homolog 1 Beta-defensin113 Endothelin-3 All-trans-retinol 13,14-reductase Lysyl oxidase homolog2 Beta-defensin 135 Low affinity immunoglobulin epsilon Regulator ofmicrotubule dynamics Long palate, lung and nasal epithelium Peptidase S1domain-containing protein Fc receptor protein 2 carcinoma-associatedprotein 4 LOC136242 Fibroblast growth factor receptor 3 R-spondin-4Lysozyme g-like protein 2 Growth/differentiation factor 7 Fibroblastgrowth factor receptor 4 Long-chain fatty acid transport protein 3Endomucin IgA-inducing protein homolog Growth arrest-specific protein 6Vesicle-trafficking protein SEC22c Neuropeptide B Putative lipocalin1-like protein 1 Growth hormone receptor Claudin-1 Kinesin-like proteinKIF7 Putative serine protease 29 Bifunctional UDP-N-acetylglucosamine 2-Leucine-rich repeats and immunoglobulin- Leukocyte-associatedimmunoglobulin- Putative scavenger receptor cysteine-richepimerase/N-acetylmannosamine kinase like domains protein 3 likereceptor 2 domain-containing protein LOC619207 Immunoglobulinsuperfamily member 8 SLAM family member 9 Calcium-dependentphospholipase A2 Secretoglobin-like protein Interleukin-4 receptor alphachain Transthyretin Proapoptotic caspase adapter protein Putativestereocilin-like protein Kallikrein-14 Serine/threonine-protein kinase32B Integrin beta-like protein 1 Insulin growth factor-like familymember 2 Kallikrein-6 Platelet-derived growth factor subunit BTolloid-like protein 1 KIR2DL4 Laminin subunit beta-3 Noggin Kunitz-typeprotease inhibitor 3 Putative zinc-alpha-2-glycoprotein-like 1Leucyl-cystinyl aminopeptidase Tryptase alpha-1 Protein TMEM155 Insulingrowth factor-like family member 4 Mannan-binding lectin serine protease1 Tetratricopeptide repeat protein 14 Prosalusin Uncharacterized proteinC2orf72 Mannan-binding lectin serine protease 2 XTP3-transactivated geneB protein Protein amnionless Replication initiation-like proteinNeutrophil gelatinase-associated lipocalin Palmitoyltransferase ZDHHC15Protein WFDC10B Prostate and testis expressed protein 3 Neuropeptide YZona pellucida sperm-binding protein 3 WAP four-disulfide core domainprotein 8 B melanoma antigen 4 Aggrecan core protein Leucine-richrepeat-containing protein 39 Protein Wnt-5b Putative uncharacterizedprotein C1orf191 Pulmonary surfactant-associated protein B Pancreatictriacylglycerol lipase Protein Wnt-7b Beta-defensin 108B-like Poliovirusreceptor-related protein 1 Transmembrane protein 139 Zonapellucida-binding protein 2 Uncharacterized protein FLJ90687 ReninLeukemia inhibitory factor SH3 domain-binding protein 5-like Secretedfrizzled-related protein 2 Ribonuclease pancreatic Galectin-1 Adipocyteadhesion molecule Basic proline-rich peptide IB-1 Semenogelin-1 C-Cmotif chemokine 21 Uncharacterized protein C12orf59 Fibroblast growthfactor 16 Signaling lymphocytic activation molecule CD5 antigen-likeApolipoprotein A-I-binding protein Serine protease inhibitor Kazal-type8 Tissue factor pathway inhibitor Carbohydrate sulfotransferase 9Claudin-17 Uncharacterized protein KIAA0495 UsherinLipopolysaccharide-binding protein Inactive caspase-12 Platelet basicprotein-like 2 Fibroblast growth factor 23 Cysteine-rich motor neuron 1protein Uncharacterized protein C7orf58 Serpin E3 Interleukin-23 subunitalpha Connective tissue growth factor Collagen alpha-1(XXVIII) chain CR1receptor Epididymal secretory protein E1 Protein eyes shut homologDentin matrix protein 4 Secreted phosphoprotein 1 ADAMTS-like protein 1Mucin-like protein 1 Uncharacterized protein C16orf48 Stress inducedsecreted protein 1 Chemokine-like factor Fibroblast growth factor 19Carboxylesterase 3 Protein Wnt EGF-like domain-containing protein 7Follistatin-related protein 3 Protein FAM20B Protein Wnt (Fragment)Tectonic-1 Hedgehog-interacting protein GPN-loop GTPase 3 Putativeserine protease LOC138652 Transmembrane protein 25 Interleukin-17receptor B GRAM domain-containing protein 1B TOM1 UDP-GalNAc:beta-1,3-N-FXYD domain-containing ion transport Phosphatidylinositol glycan anchorPutative uncharacterized protein acetylgalactosaminyltransferase 1regulator 5 biosynthesis class U protein FLJ46089 Interleukin-15 (IL-15)Endothelial lipase Interleukin-27 subunit alpha Putative uncharacterizedprotein C1orf134 Multiple epidermal growth factor-like EGF-containingfibulin-like extracellular Pro-neuregulin-4, membrane-boundUDP-GlcNAc:betaGal beta-1,3-N- domains 11 matrix protein 2 isoformacetylglucosaminyltransferase 9 Mucin and cadherin-like proteinOtoraplin Leucine-rich repeat neuronal protein 3 Uncharacterized proteinC11orf44 Ribonuclease 4 Group 3 secretory phospholipase A2 NMDAreceptor-regulated protein 2 Uncharacterized protein C12orf73 SH2domain-containing protein 3C Group XV phospholipase A2 NADH-cytochromeb5 reductase 1 Putative cystatin-9-like 2 CMP-N-acetylneuraminate-beta-Tumor necrosis factor ligand superfamily Parkinson disease 7domain-containing Putative abhydrolase domain-containinggalactosamide-alpha-2,3-sialyltransferase member 14 protein 1 proteinFAM108A5 Transmembrane protein 9 Plexin-A2 FK506-binding protein 11Beta-defensin 133 WAP four-disulfide core domain protein 2 PapilinC-type lectin domain family 12 member B Fibrosin-1 Adenosine A3 receptorProkineticin-1 Solute carrier family 35 member F5 Probable folatereceptor delta Gamma-secretase subunit APH-1A Ribonuclease 7 Sialicacid-binding Ig-like lectin 12 RPE-spondin Basigin Kunitz-type proteaseinhibitor 1 Protein FAM19A3 NPIP-like protein ENSP00000346774Baculoviral IAP repeat-containing protein 7 Spondin-2 WDrepeat-containing protein 82 Putative testis-specific prion proteinCalumenin Testican-2 Adipocyte enhancer-binding protein 1 Proline-richprotein 1 Alpha-S1-casein Inactive serine protease PAMR1 ADAMTS-likeprotein 3 Putative uncharacterized protein FP248 Cyclin-L1 Torsin-2ACoiled-coil domain-containing protein 80 UPF0670 protein C8orf55Complement factor H Vasohibin-1 Ecto-NOX disulfide-thiol exchanger 1Putative zinc-alpha-2-glycoprotein-like 2 Chorionic somatomammotropinhormone Vasorin Neuronal growth regulator 1 SPARC protein Coxsackievirusand adenovirus receptor Xylosyltransferase 1 Interphotoreceptor matrixproteoglycan 1 Otopetrin-1 Ectonucleotide Ectonucleotide cDNA FLJ36603fis, clone cDNA FLJ55667, highly similar topyrophosphatase/phosphodiesterase familypyrophosphatase/phosphodiesterase family TRACH2015180, highly similar toSecreted protein acidic and rich in member 2 member 6 Secretedfrizzled-related protein 2 cysteine ERO1-like protein alpha Oncostatin-MLipase member H Lipase member K Coagulation factor IX Derlin-1 Mucin-19(MUC-19) C-type lectin domain family 18 member C Low affinityimmunoglobulin gamma Fc HERV-FRD_6p24.1 provirus ancestral Env Psoriasissusceptibility 1 candidate gene Putative uncharacterized protein regionreceptor III-B polyprotein 2 protein UNQ6125/PRO20090 Ficolin-3Prostasin Integral membrane protein 2A Complement C3 Fc receptor-likeprotein 2 Transmembrane protease, serine 11E Vesicle transport proteinSFT2B Collagen alpha-2(IV) chain Leucine-rich repeat transmembraneprotein HLA class I histocompatibility antigen, von Willebrand factor Adomain- Uncharacterized protein FLRT3 Cw-16 alpha chain containingprotein 3A UNQ6126/PRO20091 Gelsolin Wnt inhibitory factor 1 Proteinshisa-2 homolog Serpin-like protein HMSD Granulysin C-type natriureticpeptide Signal peptidase complex subunit 3 Prostate and testis expressedprotein 4 Transmembrane glycoprotein NMB Angiopoietin-2 CD164sialomucin-like 2 protein Collagen alpha-1(XXII) chain GranulinsDeoxyribonuclease gamma Cadherin-16 Putative uncharacterized proteinC13orf28 Heparanase Carboxypeptidase A5 Cadherin-19 Cystatin-S Ig muchain C region C-C motif chemokine 14 Cerebellin-2 R-spondin-1Interleukin-1 alpha Interleukin-5 Transmembrane protein C3orf1 C8orf2Interleukin-31 receptor A Interleukin-10 Sperm equatorial segmentprotein 1 Odorant-binding protein 2a Junctional adhesion molecule BC—X—C motif chemokine 2 Uncharacterized protein C6orf72 OpiorphinLipocalin-1 C—X—C motif chemokine 5 Uncharacterized protein C11orf24Kidney androgen-regulated protein Leucine-rich repeat-containingG-protein A disintegrin and metalloproteinase with Acyl-CoA synthetasefamily member 2, Putative uncharacterized protein coupled receptor 6thrombospondin motifs 6 mitochondrial UNQ5830/PRO19650/PRO19816Latent-transforming growth factor beta- Polypeptide Probable UDP-sugartransporter protein Putative uncharacterized protein binding protein 1N-acetylgalactosaminyltransferase 1 SLC35A5 UNQ6975/PRO21958 Matrilin-3Fibulin-2 C-type lectin domain family 1 member A Tachykinin-3 Myelinprotein zero-like protein 1 Ficolin-1 C-type lectin domain family 3member A Secreted phosphoprotein 1 Neurobeachin-like protein 2 SLcytokine C-type lectin domain family 4 member E Sclerostin NicastrinFollistatin C-type lectin domain family 4 member G ADAMTS-like protein 2ADP-ribose pyrophosphatase, FRAS1-related extracellular matrix Probablecation-transporting Scavenger receptor cysteine-rich domain-mitochondrial protein 1 ATPase 13A4 containing protein LOC284297Protocadherin-15 Enamelin UPF0480 protein C15orf24 Tryptase beta-1Placenta growth factor Hyaluronan and proteoglycan link protein 1 Zonapellucida sperm-binding protein 4 Tryptase delta ProteinO-linked-mannose beta-1,2-N- Leukocyte immunoglobulin-like receptorEndoplasmic reticulum resident protein Putative cat eye syndromecritical region acetylglucosaminyltransferase 1 subfamily A member 3ERp27 protein 9 Probable hydrolase PNKD Interleukin-17F Transmembraneprotein C16orf54 Plexin domain-containing protein 1 PleiotrophinInterleukin-1 receptor accessory protein Cytochrome P450 4F12MC51L-53L-54L homolog (Fragment) Poliovirus receptor Serine proteaseinhibitor Kazal-type 5 Cytochrome P450 4X1 COBW-like placental protein(Fragment) Reticulon-4 receptor Kallikrein-15 Cytochrome P450 4Z1Cytokine receptor-like factor 2 Serum amyloid A protein Interferonalpha-14 Protein CREG2 Beta-defensin 103 Sex hormone-binding globulinPregnancy-specific beta-1-glycoprotein 4 DnaJ homolog subfamily B member9 Beta-defensin 106 SLAM family member 6 Collagenase 3 Dipeptidase 3Hyaluronidase-3 Sarcolemmal membrane-associated protein Matrixmetalloproteinase-16 Membrane protein FAM174A Interleukin-28 receptoralpha chain Sushi, von Willebrand factor type A, EGF Pituitary adenylatecyclase-activating Thioredoxin domain-containing Glycosyltransferase 54domain-containing and pentraxin domain-containing protein 1 polypeptideprotein 15 protein Thyroxine-binding globulin Prokineticin-2 ProteinFAM19A4 Chordin-like protein 1 Transmembrane and coiled-coil domain-Latent-transforming growth factor beta- Adenosine monophosphate-proteinPutative uncharacterized protein containing protein 1 binding protein 3transferase FICD UNQ9370/PRO34162 Transmembrane protease, serine 3Somatoliberin Prenylcysteine oxidase-like Netrin receptor UNC5B Tumornecrosis factor receptor superfamily Thrombospondin type-1domain-containing Phytanoyl-CoA hydroxylase-interacting Fibroblastgrowth factor receptor FGFR-1 member 10C protein 1 protein-like secretedform protein (Fragment) Tumor necrosis factor receptor superfamilyAngiogenic factor with G patch and FHA FXYD domain-containing iontransport Uncharacterized protein member 11B domains 1 regulator 4ENSP00000244321 Serotransferrin TGF-beta receptor type IIIGrowth/differentiation factor 11 ECE2 Tryptase beta-2 Thyrotropinsubunit beta Cerebral dopamine neurotrophic factor EPA6 Protein YIPF5Uncharacterized protein C19orf36 GPN-loop GTPase 2 Putative solubleinterleukin 18 receptor 1 Vesicle-associated membrane protein-Complement C1q tumor necrosis Growth hormone-inducible Putativeabhydrolase domain-containing associated protein B/C factor-relatedprotein 2 transmembrane protein protein FAM108A6 cDNA, FLJ96669, highlysimilar to Homo Ectonucleotide Glycerophosphodiester Putative V-set andimmunoglobulin sapiens secreted protein, acidic, cysteine-pyrophosphatase/phosphodiesterase family phosphodiesterasedomain-containing domain-containing-like protein rich(osteonectin)(SPARC), mRNA member 5 protein 2 ENSP00000303034 cDNAFLJ77519, highly similar to Homo Polypeptide N- WAP, kazal,immunoglobulin, kunitz and B cell maturation antigen transcript variantsapiens secreted frizzled related proteinacetylgalactosaminyltransferase-like NTR domain-containing protein 1 4(Tumor necrosis factor receptor mRNA protein 2 superfamily member 17)T-cell differentiation antigen CD6 Slit homolog 1 protein KDELmotif-containing protein 1 UPF0672 protein C3orf58 Pikachurin Growthhormone variant Adipophilin Methylthioribose-1-phosphate isomeraseFibrinogen-like protein 1 Angiopoietin-related protein 3 Lactase-likeprotein 17-beta hydroxysteroid dehydrogenase 13 Interleukin-32Angiopoietin-related protein 7 Chondromodulin-1 Aminopeptidase BMatrilin-4 Ecto-ADP-ribosyltransferase 5 Collagen alpha-6(VI) chainDermcidin Sperm-associated antigen 11B Carbonic anhydrase-relatedprotein 11 Leucine-rich repeat-containing protein 33 MeteorinCoagulation factor XII Probable ribonuclease 11 MANSC domain-containingprotein 1 Methyltransferase-like protein 7A Hepcidin Probablecarboxypeptidase X1 Lipocalin-15 NL3 Klotho Protein FAM3D ArylsulfataseI N-acetyltransferase 15 Serglycin C—X—C motif chemokine 14 Mesodermdevelopment candidate 2 Ephrin-A4 Tomoregulin-2 Beta-defensin 127Dickkopf-related protein 1 Protein Plunc Chordin-like protein 2Beta-defensin 129 Podocan Kallikrein-11 Tumor necrosis factor receptorsuperfamily Cysteine-rich secretory protein LCCL Fibronectin type IIIdomain-containing WNT1 induced secreted protein 1 splice member 6Bdomain-containing 2 protein 1 variant x (Fragment) UPF0414 transmembraneprotein C20orf30 Fibroblast growth factor 21 Neurotrimin Interleukin-1family member 10 C-type lectin domain family 4 member C Plasmaalpha-L-fucosidase Olfactory receptor 10W1 PLA2G2D UPF0317 proteinC14orf159, mitochondrial Gastrokine-1 Protein PARM-1 Proteoglycan 3Netrin-G2 Gastrokine-2 PDZ domain-containing protein 2 Insulin-likepeptide INSL5 Metalloreductase STEAP2 Glutathione peroxidase 7Proepiregulin Olfactomedin-like protein 3 Sushi domain-containingprotein 4 HHIP-like protein 1 Polycystic kidney disease protein 1-like 1Extracellular glycoprotein lacritin Protein YIF1B Interferon kappaWLPL514 Retinol dehydrogenase 13 Apolipoprotein M Apolipoprotein C-IMatrix metalloproteinase-26 Neutrophil defensin 3 C4b-binding proteinbeta chain Procollagen C-endopeptidase enhancer 2 RELT-like protein 2GLGQ5807 T-cell surface glycoprotein CD8 beta chain Left-rightdetermination factor 1 Solute carrier family 35 member E3 TUFT1 C-Cmotif chemokine 3-like 1 Leucine-rich repeat LGI family member 4 Zinctransporter ZIP9 DRLV8200 Fibroblast growth factor 8 BRCA1-A complexsubunit Abraxas Noelin-2 IDLW5808 Sialomucin core protein 24 Leucinezipper protein 2 Seizure 6-like protein 2 UBAP2 Programmed cell death 1ligand 2 Neurexophilin-3 Semaphorin-3A C1q/TNF-related protein 8Secreted and transmembrane 1 Osteomodulin Semaphorin-4C KIR2DL4(Fragment) Complement C1q tumor necrosis factor- Kazal-type serineprotease inhibitor Abhydrolase domain-containing protein Chemokine-likefactor super family 2 related protein 6 domain-containing protein 1 14Atranscript variant 2 EGF-like module-containing mucin-like Spermacrosome membrane-associated Ankyrin repeat domain-containingKeratinocytes associated transmembrane hormone receptor-like 3 protein 3protein 36 protein 1 Noelin-3 Secretoglobin family 3A member 1 Proteinshisa-4 GKGM353 Odorant-binding protein 2b Tsukushin Neuromedin-UMATL2963 Urotensin-2 Claudin-2 (SP82) Nodal homolog NINP6167 VitrinComplement factor H-related protein 2 Synaptogyrin-2 POM121-likeWNT1-inducible-signaling pathway Immunoglobulin superfamily containingBrain-specific angiogenesis inhibitor 1- RTFV9368 (SLE-dependent protein3 leucine-rich repeat protein associated protein 2-like protein 2upregulation 1) cDNA FLJ75759, highly similar to Homo Leucine-richrepeat and immunoglobulin- Coiled-coil domain-containing Leucine-richrepeat and immunoglobulin- sapiens follistatin-like 3 (secreted likedomain-containing nogo receptor- protein 104 like domain-containing nogoreceptor- glycoprotein) (FSTL3), mRNA interacting protein 1 interactingprotein 4 Angiotensin-converting enzyme 2 Kin of IRRE-like protein 3Transmembrane 4 L6 family member 20 KCNQ2 Adiponectin Hematopoietic cellsignal transducer Transmembrane protein 107 ELCV5929Angiopoietin-related protein 4 Follitropin subunit beta Transmembraneprotein 143 KVVM3106 Apolipoprotein A-V Melanoma inhibitory activityprotein 3 Transmembrane protein 178 ISPF6484 Asporin Leucine-richrepeat-containing protein 4 Transmembrane protein 205 LKHP9428Bactericidal permeability-increasing protein Zinc transporter 5Transmembrane protein 41A VNFT9373 CUB domain-containing protein 1Leucine-rich repeat neuronal protein 1 Transmembrane protein 50AACAH3104 Cartilage intermediate layer protein 1 Apical endosomalglycoprotein Transmembrane protein 50B RVLA1944 Beta-Ala-His dipeptidaseSerum amyloid A-4 protein Interleukin-28B Wpep3002 Collagen alpha-1(V)chain Probetacellulin Neuronal pentraxin-2 ZDHHC11 Collagen alpha-1(XXV)chain Beta-1,4-galactosyltransferase 7 Collectrin AGLW2560 Estradiol17-beta-dehydrogenase 11 3-hydroxybutyrate dehydrogenase type 2Transmembrane protein 92 TSSP3028 DnaJ homolog subfamily C member 10C1GALT1-specific chaperone 1 Transmembrane protein 95 RFVG5814 EGF-likedomain-containing protein 6 Beta-casein Transmembrane protein 9BSHSS3124 Coagulation factor XIII A chain Kappa-casein Probablecarboxypeptidase PM20D1 MMP19 Glucose-6-phosphate isomeraseTransmembrane protein C2orf18 Tetraspanin-12 GSQS6193Appetite-regulating hormone Carboxypeptidase N catalytic chainTetraspanin-13 VGPW2523 Interleukin-12 subunit beta CD320 antigenTetraspanin-15 LMNE6487 Interleukin-22 Chondroitin sulfate synthase 1UPF0513 transmembrane protein ALLA2487 Intelectin-1 Chondroitin sulfatesynthase 2 Mitochondrial uncoupling protein 4 GALI1870 Leucine-richglioma-inactivated protein 1 CMRF35-like molecule 7 Polyserase-2FRSS1829 Lymphocyte antigen 96 Protein canopy homolog 3 Probablepalmitoyltransferase ZDHHC24 MRSS6228 Matrilysin Short-chaindehydrogenase/reductase 3 Zona pellucida sperm-binding protein 1GRPR5811 Mucin-20 Delta-like protein 4 Zona pellucida sperm-bindingprotein 2 AVLL5809 Proprotein convertase subtilisin/kexin Delta andNotch-like epidermal growth Conserved oligomeric Golgi complex CR1C3b/C4b receptor SCR9 (or 16) C- type 9 factor-related receptor subunit7 term. exon SCR = short consensus repeat Peptidoglycan recognitionprotein Dolichol kinase Adiponectin receptor protein 2 PIKR2786Interferon-induced 17 kDa protein Endothelin-converting enzyme-like 1Inhibin beta C chain S100 calcium binding protein A7-like 3 ProteinWnt-4 Integral membrane protein 2B Brorin GTWW5826 (LP5085 protein)Allograft inflammatory factor 1-like Insulin-like growth factor-bindingprotein 5 Semaphorin-3C KTIS8219 (HCG2020043) Armadillorepeat-containing X-linked Endothelial cell-selective adhesion Heparansulfate glucosamine 3-O- Hyaluronan and proteoglycan link protein 3molecule sulfotransferase 2 protein 4 Chondroitin sulfate N- Signalpeptide, CUB and EGF-like domain- Leptin receptor overlappingtranscript- Micronovel acetylgalactosaminyltransferase 1 containingprotein 1 like 1 Chitotriosidase-1 Complement factor H-related protein 3SPARC-like protein 1 SAMK3000 Claudin domain-containing protein 1Prorelaxin H1 Fibulin-7 VFLL3057 Erlin-2 Follistatin-related protein 1Protein HEG homolog 1 CVWG5837 Glycosyltransferase 8 domain-containingGloboside alpha-1,3-N- Fibrinogen C domain-containing VGSA5840 protein 1acetylgalactosaminyltransferase 1 protein 1 Golgi membrane protein 1Gamma-glutamyl hydrolase Phospholipase A1 member A GHPS3125 ProbableG-protein coupled receptor 125 Cadherin-24 Basic salivary proline-richprotein 2 GRTR3118 Interleukin-20 receptor alpha chainGlycerol-3-phosphate acyltransferase 3 Spermatogenesis-associatedprotein 6 PAMP6501 Galectin-7 G-protein coupled receptor 56 Sushirepeat-containing protein SRPX2 LTLL9335 NKG2D ligand 4Hyaluronan-binding protein 2 Twisted gastrulation protein homolog 1VCEW9374 L-amino-acid oxidase Proheparin-binding EGF-like growth factorTorsin-1B AHPA9419 Prolyl 3-hydroxylase 1 Histidine-rich glycoproteinProtein Wnt-5a MDHV1887 GPI ethanolamine phosphate transferase 2Carbohydrate sulfotransferase 14 Acrosin-binding protein HSAL5836 GPIethanolamine phosphate transferase 3 Interleukin-20 receptor beta chainC-type lectin domain family 18 member B LHLC1946 Calcium-bindingmitochondrial carrier Ectonucleotide Lysosomal-associated transmembraneLong palate, lung and nasal epithelium protein SCaMC-2 (Smallcalcium-binding pyrophosphatase/phosphodiesterase protein 4Acarcinoma-associated protein 3 (Ligand- mitochondrial carrier protein 2)family member 3 binding protein RYA3) Pulmonary surfactant-associatedprotein A2 Insulin-like growth factor-binding protein 7 Semaphorin-3ELPPA601 Splicing factor, arginine/serine-rich 16 KallistatinAmeloblastin PINK1 Alpha-N-acetylgalactosaminide alpha-2,6- Fibronectintype III domain-containing Major facilitator superfamily domain-SERH2790 sialyltransferase 6 protein 3B containing protein 5 Single IgIL-1-related receptor Leukemia inhibitory factor receptor Angiopoietin-1FLFF9364 Tectonic-3 Lin-7 homolog B Angiopoietin-4 APELIN Tumor necrosisfactor ligand superfamily Thioredoxin-related transmembrane Multipleepidermal growth factor-like GLSH6409 member 11 protein 1 domains 9Tumor necrosis factor receptor superfamily Disintegrin andmetalloproteinase domain- Acid sphingomyelinase-like SFVP2550 member 19containing protein 32 phosphodiesterase 3a Palmitoyltransferase ZDHHC9Ly6/PLAUR domain-containing protein 3 ADAMTS-like protein 5 RRLF9220Fibulin-5 C-type lectin domain family 14 member A Spexin PTML5838Protein Z-dependent protease inhibitor Protein cornichon homologPutative trypsin-6 VLGN1945 Alpha-2-macroglobulin Protein FAM151AProto-oncogene protein Wnt-1 AVPC1948 Agouti-related proteinFK506-binding protein 14 Bone morphogenetic protein 3b AWQG2491Pancreatic alpha-amylase Neuropilin and tolloid-like protein 2 Bonemorphogenetic protein 5 PSVL6168 Natriuretic peptides B Protocadherinbeta-13 Bone morphogenetic protein 8B LCII3035 Atrial natriuretic factorPrenylcysteine oxidase 1 Protein FAM26D PPRR6495 Neutral ceramidasePeflin C1q-related factor RLSC6348 Beta-2-microglobulin Peptidyl-prolylcis-trans isomerase-like 1 WAP four-disulfide core domain protein 1CSRP2BP Bone morphogenetic protein 4 Prostate stem cell antigenCerebellin-1 GLLV3061 Biotinidase Protein patched homolog 2Carboxypeptidase O GWSI6489 Scavenger receptor cysteine-rich type 1Chitobiosyldiphosphodolichol beta- Myelin protein zero-like protein 2cDNA FLJ53955, highly similar to protein M130 mannosyltransferase(Epithelial V-like antigen 1) Secreted frizzled-related protein 4Carboxypeptidase B2 Protein sel-1 homolog 1 Serine protease 1-likeprotein 1 PPIF Carboxypeptidase Z ProSAAS Coiled-coil domain-containingprotein 70 VSSW1971 C-C motif chemokine 5 Sialic acid-binding Ig-likelectin 9 C-C motif chemokine 28 KLIA6249 C-C motif chemokine 7 SLIT andNTRK-like protein 1 Uncharacterized protein C4orf29 ALLW1950 C-C motifchemokine 8 Statherin CUB domain-containing protein 2 GVEI466 CD59glycoprotein Testisin Trem-like transcript 4 protein ESFI5812 Complementfactor I Transmembrane channel-like protein 5 Uncharacterized proteinC6orf58 GNNC2999 Clusterin Transmembrane protease, serine 4Chondroadherin AAGG6488 Collagen alpha-2(I) chain Metastasis-suppressorKiSS-1 Cartilage intermediate layer protein 2 HHSL751 Collagenalpha-1(III) chain Islet amyloid polypeptide Uncharacterized proteinC10orf25 Beta-defensin 108B Collagen alpha-1(IV) chain Trem-liketranscript 2 protein Isthmin-1 Beta-defensin 118 Collagen alpha-3(IV)chain Thioredoxin domain-containing protein 12 Cystatin-8 Beta-defensin124 Collagen alpha-5(IV) chain Vascular endothelial growth factor BCardiotrophin-1 (CT-1) Beta-defensin 125 Collagen alpha-3(VI) chainVascular endothelial growth factor C Chymotrypsinogen B Beta-defensin126 Complement component C6 Reticulocalbin-3 C—X—C motif chemokine 9Deoxyribonuclease-1-like 2 Collagen alpha-1(IX) chain Fibrillin-1 C—X—Cmotif chemokine 13 Stanniocalcin-2 Collagen alpha-1(X) chain ProteinFAM3A EMILIN-3 Endothelial cell-specific molecule 1 Collagenalpha-1(XVII) chain Protein G7c Secretagogin Carboxylesterase 7 Collagenalpha-1(XXI) chain Neuropilin and tolloid-like protein 1 Epididymalsecretory protein E3-alpha Protein NOV homolog Coatomer subunit alphaPregnancy-specific beta-1-glycoprotein 11 Epiphycan UPF0528 proteinFAM172A Complement receptor type 1 Serpin B4 Protein FAM5CInterleukin-27 subunit beta Cystatin-SN ADAM DEC1 Fibroblast growthfactor 20 Protein FAM3C Deoxyribonuclease-1 ADP-dependent glucokinaseFibroblast growth factor-binding protein 3 Stromal cell-derived factor2-like protein 1 Extracellular matrix protein 1 Alpha-amylase 2BTransmembrane protein 204 Butyrophilin subfamily 1 member A1 Lowaffinity immunoglobulin gamma UDP-GlcNAc:betaGal beta-1,3-N-Phosphatidylethanolamine-binding Keratinocyte-associated transmembraneFc region receptor III-A acetylglucosaminyltransferase 3 protein 4protein 2 Alpha-fetoprotein Calcitonin gene-related peptide 2Coagulation factor V Immunoglobulin alpha Fc receptor Heparin-bindinggrowth factor 2 Carboxypeptidase E Coagulation factor VII EMILIN-2Fibrinogen gamma chain Cardiotrophin-like cytokine factor 1 Pro-MCHEphrin type-A receptor 10 Growth/differentiation factor 5 Collagenalpha-2(VIII) chain Folate receptor gamma Exostosin-like 2 Glial cellline-derived neurotrophic factor Crumbs homolog 2 Mucin-7Follistatin-related protein 4 Insulin-like growth factor-binding protein3 Dentin matrix acidic phosphoprotein 1 Galanin-like peptideFollistatin-related protein 5 Insulin-like growth factor IA Downsyndrome cell adhesion molecule Hemicentin-1 Transmembrane protein 66 Iggamma-1 chain C region Immunoglobulin superfamily member 1 Interleukin-6Growth/differentiation factor 2 Ig gamma-2 chain C region Interleukin-4Embryonic growth/differentiation factor 1 GDNF family receptor alpha-4Ig gamma-3 chain C region Interleukin-6 receptor subunit alphaInterleukin-8 Ig gamma-4 chain C region Insulin-like 3 Interleukin-24Gremlin-2 Lymphocyte antigen 86 Inter-alpha-trypsin inhibitor heavychain Ladinin-1 Stromelysin-2 Inhibin beta E chain UPF0378 proteinKIAA0100 Lipase member I Probable G-protein coupled receptor 171 GRAMdomain-containing protein 1C Kininogen-1 Pancreatic lipase-relatedprotein 1 Pappalysin-2 Interferon alpha-10 Laminin subunit alpha-2Leucine-rich alpha-2-glycoprotein Microfibril-associated glycoprotein 4Interferon alpha-16 Laminin subunit alpha-4 Matrix-remodeling-associatedprotein 5 Neuromedin-B Interferon alpha-6 Laminin subunit beta-1Netrin-4 Mimecan Immunoglobulin superfamily member 21 Protein-lysine6-oxidase Hepatocyte growth factor receptor Matrix metalloproteinase-19Agrin Multimerin-1 C-C motif chemokine 22 Interleukin-11 ProlactinVasopressin-neurophysin 2-copeptin Nyctalopin Interleukin-17A Kelch-likeprotein 11 Nidogen-1 Osteocalcin Interleukin-18 Protein Wnt-16Phospholipase A2, Basic salivary proline-rich protein 3 Interleukin-26Properdin Perforin-1 Pregnancy-specific beta-1-glycoprotein 10Interleukin-28A Kallikrein-13 Phosphatidylinositol-glycan-specificLeucine-rich repeat transmembrane protein Transmembrane emp24 domain-1-acyl-sn-glycerol-3-phosphate phospholipase D FLRT2 containing protein3 acyltransferase delta Fibrocystin R-spondin-3 Interleukin-29Kallikrein-9 Phospholipid transfer protein Sialoadhesin Insulin-likepeptide INSL6 Vitamin K-dependent protein S Prostatic acid phosphataseTrypsin-3 Protein Wnt-2b Butyrophilin-like protein 8 Vitamin K-dependentprotein Z Dipeptidase 2 Pregnancy-specific beta-1-glycoprotein 1 Lamininsubunit beta-4 Salivary acidic proline-rich Collagen and calcium-bindingEGF Sperm acrosome membrane-associated Lymphatic vessel endothelialhyaluronic phosphoprotein 1/2 domain-containing protein 1 protein 4 acidreceptor 1 Pregnancy zone protein Germ cell-specific gene 1-like proteinLaminin subunit gamma-3 Cystatin-SA Prorelaxin H2 Leucine-richrepeat-containing protein 31 Lysyl oxidase homolog 3 Transmembraneprotein 59 Semaphorin-4D Apolipoprotein O Neurotensin/neuromedin NApolipoprotein(a)-like protein 2 Slit homolog 2 protein Dystroglycan MAMdomain-containing protein 2 Lysozyme-like protein 2 Alpha-tectorinNeutrophil defensin 4 Microfibrillar-associated protein 2 Lysozyme-likeprotein 4 Tenascin-X Amphoterin-induced protein 3 Melanoma inhibitoryactivity protein 2 Reelin Trefoil factor 3 Gamma-secretase subunitAPH-1B Matrix metalloproteinase-24 Retinol-binding protein 4 Transferrinreceptor protein 1 Apolipoprotein C-IV Matrix metalloproteinase-25Carbonic anhydrase 14 Protransforming growth factor alpha ArylsulfataseG Netrin-1 Tubulointerstitial nephritis antigen Transforming growthfactor beta-2 Glia-activating factor Netrin-3 Neuropeptide W Tumornecrosis factor ligand superfamily Caspase recruitment domain-containingAlpha-N-acetylgalactosaminide alpha- Alpha-1,3-mannosyl-glycoprotein4-beta- member 6 protein 18 2,6-sialyltransferase 1N-acetylglucosaminyltransferase B Tumor necrosis factor receptorsuperfamily Heparan sulfate glucosamine 3-O-Alpha-N-acetylgalactosaminide alpha- Transmembrane emp24 domain- member1B sulfotransferase 3A1 2,6-sialyltransferase 3 containing protein 5Tumor necrosis factor receptor superfamily Thyrotropin-releasinghormone-degrading Melanoma-derived growth regulatory Complement C1qtumor necrosis factor- member 5 ectoenzyme protein related protein 3Thrombopoietin Guanylin FMRFamide-related peptides Podocan-like protein1 VIP peptides Choline transporter-like protein 3 Otoconin-90Pregnancy-specific beta-1-glycoprotein 5 Acidic mammalian chitinase17-beta-hydroxysteroid dehydrogenase 14 Neurturin KeratocanCysteine-rich secretory protein 2 Immunoglobulin lambda-like polypeptide1 Neurexophilin-1 Group IIE secretory phospholipase A2Haptoglobin-related protein DnaJ homolog subfamily B member 14Neurexophilin-2 Left-right determination factor 2 C-C motif chemokine 26F-box only protein 8 Platelet factor 4 variant NKG2D ligand 2Collectin-11 Fibroleukin Nociceptin Macrophage metalloelastaseCysteine-rich with EGF-like domain Methionine-R-sulfoxide reductase B3,V-set and transmembrane domain- Triggering receptor expressed on myeloidprotein 2 mitochondrial containing protein 1 cells 1 C—X—C motifchemokine 16 Leucine-rich repeat LGI family member 2 Proline-richprotein 4 Cytokine receptor-like factor 1 Fibroblast growthfactor-binding protein 1 Vesicle transport protein GOT1BProlactin-releasing peptide Secretin Interleukin-1 family member 5Integral membrane protein GPR177 Serine protease 33 Stromal cell-derivedfactor 2 Interleukin-1 family member 9 Probable G-protein coupledreceptor 78 Pregnancy-specific beta-1-glycoprotein 8 Lysozyme-likeprotein 6 Kallikrein-5 HEPACAM family member 2 Retbindin Serpin A9Matrilin-2 Interleukin-27 receptor subunit alpha FMRFamide-relatedpeptides Sclerostin domain-containing protein 1 Cell surfaceglycoprotein CD200 receptor 1 Proenkephalin-A Ribonuclease K6Lysocardiolipin acyltransferase 1 Lysophosphatidic acid phosphatase type6 Integrin alpha-10 Ribonuclease T2 Plasma glutamate carboxypeptidaseNucleotide exchange factor SIL1 KTEL motif-containing protein 1 RepetinSlit homolog 3 protein Thrombospondin type-1 domain-containing Leukocyteimmunoglobulin-like receptor Complement C1r subcomponent-like C3 andPZP-like alpha-2-macroglobulin protein 4 subfamily A member 5 proteindomain-containing protein 8 WNT1-inducible-signaling pathway protein 2Leucine-rich repeat and fibronectin type-III Uncharacterizedglycosyltransferase Retinoic acid receptor responder domain-containingprotein 3 AER61 protein 2 Bromodomain-containing protein 9 UteroglobinSemaphorin-3G Cartilage acidic protein 1 CD99 antigen-like protein 2Netrin-G1 ligand Secretoglobin family 1C member 1 Stanniocalcin-1Uncharacterized protein C1orf159 Pannexin-1 Secretoglobin family 1Dmember 1 Beta-tectorin Carbohydrate sulfotransferase 12 Protocadherin-12Secretoglobin family 1D member 2 Post-GPI attachment to proteins factor3 Probable serine carboxypeptidase CPVL Protocadherin alpha-10 SerpinA12 Germ cell-specific gene 1 protein Mucin-3A Protocadherin beta-10Serpin I2 Interleukin-21 receptor CUB and zona pellucida-like domain-Osteopetrosis-associated transmembrane von Willebrand factor C and EGFV-set and immunoglobulin domain- containing protein 1 protein 1domain-containing protein containing protein 4 Polypeptide N-Beta-galactoside alpha-2,6- A disintegrin and metalloproteinase withScavenger receptor cysteine-rich domain- acetylgalactosaminyltransferase14 sialyltransferase 1 thrombospondin motifs 15 containing group Bprotein Galectin-9 GPI transamidase component PIG-S Sodium channelsubunit beta-2 Prothyroliberin Leucine-rich repeat-containing protein 17Proline-rich transmembrane protein 3 Metalloproteinase inhibitor 4Semaphorin-4A Leucine-rich repeat neuronal protein 2 Sulfhydryl oxidase2 T-cell immunomodulatory protein Bifunctional heparan sulfate N- Adisintegrin and metalloproteinase with A disintegrin andmetalloproteinase with Tumor necrosis factor receptordeacetylase/N-sulfotransferase 3 thrombospondin motifs 16 thrombospondinmotifs 10 superfamily member 27 Tuftelin SH2 domain-containing protein3A Thymic stromal lymphopoietin Toll-like receptor 7 Brain mitochondrialcarrier protein SHC-transforming protein 4 Transmembrane protein 130Signal peptide, CUB and EGF-like domain- Disintegrin andmetalloproteinase domain- Unique cartilage matrix-associated Thioredoxindomain-containing containing protein 3 containing protein 23 proteinprotein 16 14-3-3 protein sigma Transducin beta-like protein 2Urocortin-2 Alpha-2-antiplasmin Alpha-1-acid glycoprotein 1 Tudordomain-containing protein 10 Urocortin-3 ( WAP four-disulfide coredomain protein 3 Alpha-1-acid glycoprotein 2 Transmembrane 9 superfamilymember 3 Protein AMBP Protein WFDC9 von Willebrand factor Adomain-containing Von Willebrand factor D and EGF domain- Complement C1qtumor necrosis factor- A disintegrin and metalloproteinase with protein1 containing protein related protein 9-like thrombospondin motifs 14Disintegrin and metalloproteinase domain- A disintegrin andmetalloproteinase with Growth inhibition and differentiation- Adipocyteplasma membrane-associated containing protein 9 thrombospondin motifs 17related protein 88 protein Angiotensinogen Transmembrane channel-likeprotein 2 Protein Wnt-10a Peroxidasin homolog Apolipoprotein A-II(Apo-AII) (ApoA-II) Pregnancy-specific beta-1-glycoprotein 3 ProteinWnt-3a Progressive ankylosis protein homolog Apolipoprotein A-IV(Apo-AIV) (ApoA-IV) Tenomodulin Proto-oncogene protein Wnt-3Chitinase-3-like protein 1 Apolipoprotein C-II (Apo-CII) (ApoC-II)Tetraspanin-6 Protein Wnt-6 UPF0672 protein CXorf36 Beta-2-glycoprotein1 Thioredoxin domain-containing protein 5 Protein Wnt-9a Arylsulfatase JApoptosis-related protein 3 Vascular endothelial growth factor DCytokine SCM-1 beta Cortistatin Beta-secretase 2 Pregnancy-specificbeta-1-glycoprotein 9 Zymogen granule membrane protein 16 CeruloplasminHisto-blood group ABO system transferase Semaphorin-3F Zonapellucida-binding protein 1 Angiopoietin-related protein 5 Cathepsin L2Acid phosphatase-like protein 2 Anterior gradient protein 3 homologCoiled-coil domain-containing protein 126 C-C motif chemokine 3Apolipoprotein O-like Amelotin CD177 antigen C-type lectin domain family1 member B Beta-defensin 119 Uncharacterized protein C5orf46 Proteincanopy homolog 4 Calcium-activated chloride channel A disintegrin andmetalloproteinase with Uncharacterized aarF domain-containingFibronectin type-III domain-containing regulator 1 thrombospondin motifs12 protein kinase 1 protein C4orf31 Chymase Protein FAM131A DraxinProtein FAM180A Collagen alpha-1(VI) chain Protein FAM3B Fibroblastgrowth factor 18 Platelet basic protein Complement component C8 alphachain Beta-galactosidase-1-like protein C—X—C motif chemokine 11Interferon epsilon Complement component C9 Lysozyme g-like protein 1Ly6/PLAUR domain-containing protein 6 Intelectin-2 Glucose-fructoseoxidoreductase domain- Inter-alpha-trypsin inhibitor heavy chainChymotrypsin-like elastase family Alpha-1,3-mannosyl-glycoprotein4-beta- containing protein 2 H5-like protein member 1N-acetylglucosaminyltransferase A DnaJ homolog subfamily B member 11Sperm acrosome-associated protein 5 Erythropoietin receptor Matrixextracellular phosphoglycoprotein Ectonucleotide Leucine-rich repeat andimmunoglobulin- MAM domain-containing cDNA FLJ77863, highly similar toHomo pyrophosphatase/phosphodiesterase family like domain-containingnogo receptor- glycosylphosphatidylinositol anchor sapiens secreted andtransmembrane 1 member 7 interacting protein 2 protein 2 (SECTM1), mRNAEndoplasmic reticulum aminopeptidase 1 Surfactant-associated protein 2Matrix metalloproteinase-27 Epididymal-specific lipocalin-6 Receptortyrosine-protein kinase erbB-3 Adiponectin receptor protein 1 Inactiveserine protease 35 Afamin Endoplasmic reticulum resident proteinMultiple epidermal growth factor-like Coiled-coil domain-containingProbable cation-transporting ATPase ERp44 domains 6 protein 134 13A5IgGFc-binding protein Neuroendocrine protein 7B2 Suprabasin Glutathioneperoxidase 3 Complement factor H-related protein 1 Alpha-1B-glycoproteinSecretoglobin family 1D member 4 Claudin-18 Polypeptide N- WAP, kazal,immunoglobulin, kunitz and V-set and transmembrane domain- Putativekiller cell immunoglobulin-like acetylgalactosaminyltransferase 2 NTRdomain-containing protein 2 containing protein 2A receptor like proteinKIR3DP1 Hemopexin Arylacetamide deacetylase-like 1 ADM Secretoryphospholipase A2 receptor Hepatocyte growth factor activator Histatin-3Uncharacterized protein C2orf82 Haptoglobin Major histocompatibilitycomplex class I- Pro-neuregulin-3, membrane-bound Insulin growthfactor-like family Carcinoembryonic antigen-related cell related geneprotein isoform member 1 adhesion molecule 20 Insulin-like growthfactor-binding protein 6 Agouti-signaling protein Cadherin-like protein29 Bone morphogenetic protein 3 Ig delta chain C region Claudin-8 Bonemorphogenetic protein 15 Bone marrow stromal antigen 2 Interleukin-1beta UPF0454 protein C12orf49 Plasma serine protease inhibitorCytochrome P450 20A1 Low-density lipoprotein receptor-related vonWillebrand factor A domain-containing Carcinoembryonic antigen-relatedcell Bactericidal/permeability-increasing protein 10 protein 5B1adhesion molecule 21 protein-like 3 Junctional adhesion molecule CCadherin-6 Alpha-lactalbumin Protein dpy-19 homolog 2 Uncharacterizedprotein KIAA0319 Cathelicidin antimicrobial peptide Sister chromatidcohesion protein DCC1 Group IIF secretory phospholipase A2 Lamininsubunit alpha-5 Laminin subunit gamma-1 Galectin-3-binding proteinCarboxypeptidase B Fibronectin type III domain-containingDehydrogenase/reductase SDR family Dynein heavy chain domain-containingGlycosyltransferase 8 domain-containing protein 4 member 7B protein 1protein 2 Lipoprotein lipase C-C motif chemokine 16 C-C motif chemokine17 Protein FAM19A1 Interstitial collagenase C-C motif chemokine 24 Fattyacyl-CoA reductase 1 GDNF family receptor alpha-like Matrixmetalloproteinase-9 HEAT repeat-containing protein C7orf27 Fin budinitiation factor homolog Probable glutathione peroxidase 8 Mucin-16Collagen alpha-2(IX) chain Polymeric immunoglobulin receptor Cystatin-DMucin-2 Collagen alpha-3(IX) chain Prion-like protein doppel Cystatin-FMucin-5B Colipase C—X—C motif chemokine 6 Platelet-activating factoracetylhydrolase Myocilin Collagen alpha-1(XXVII) chain C—X—C motifchemokine 10 Pappalysin-1 Oxidized low-density lipoprotein receptor 1Carboxypeptidase N subunit 2 Beta-defensin 1 Solute carrier family 22member 12 Prostate tumor overexpressed gene 1 Leucine-rich repeattransmembrane Hyaluronan and proteoglycan link Chorionicsomatomammotropin hormone- protein neuronal protein 4 protein 2 like 1Receptor-interacting serine/threonine- Collagen triple helixrepeat-containing Disintegrin and metalloproteinase Regulator ofmicrotubule dynamics protein kinase 2 protein 1 domain-containingprotein 30 protein 3 Equilibrative nucleoside transporter 3 Endothelin-2Suppressor of fused homolog Retinol dehydrogenase 14 Selenoprotein PFibromodulin Folate receptor beta Galanin Pulmonarysurfactant-associated protein D Fc receptor-like B Extracellularsulfatase Sulf-2 Transcobalamin-2 Stimulated by retinoic acid gene 6protein Zinc finger RAD18 domain-containing Tumor necrosis factorreceptor Catechol-O-methyltransferase domain- homolog protein C1orf124superfamily member 14 containing protein 1 Trefoil factor 1Growth/differentiation factor 15 Artemin Tripeptidyl-peptidase 1 Tissuefactor pathway inhibitor 2 Glia-derived nexin Collagen alpha-1(XII)chain Trem-like transcript 1 protein Prothrombin Progonadoliberin-1Collagen alpha-1(XIV) chain Guanylate cyclase activator 2B Toll-likereceptor 9 Granzyme K Beta-defensin 2 Inducible T-cell costimulatorIntercellular adhesion molecule 4 Interferon alpha-17 Interleukin-21Interleukin-19 Interferon alpha-21 Interleukin-3 Isthmin-2 Interferonalpha-8 Interleukin-7 Notch homolog 2 N-terminal-like protein Kin ofIRRE-like protein 1 Interferon omega-1 Inhibin alpha chain Lamininsubunit beta-2 Kallikrein-10 Early placenta insulin-like peptide Lamininsubunit alpha-3 Neuropilin-2 Latent-transforming growth factor beta-EGF, latrophilin and seven transmembrane Dehydrogenase/reductase SDRfamily EGF-containing fibulin-like extracellular binding protein 4domain-containing protein 1 member on chromosome X matrix protein 1Paired immunoglobulin-like type 2 receptor Fibronectin type 3 andankyrin repeat FXYD domain-containing ion transport Receptor-typetyrosine-protein alpha domains protein 1 regulator 6 phosphatase kappaRegenerating islet-derived protein 3 alpha Lysyl oxidase homolog 4Serine incorporator 2 Regenerating islet-derived protein 4 E3ubiquitin-protein ligase RNF5 Lumican Stromelysin-3 Tachykinin-4Protachykinin-1 Adropin Secreted phosphoprotein 1 Matrixmetalloproteinase-23 Secreted frizzled-related protein 1, isoformLeucine-rich repeat transmembrane protein Serine beta-lactamase-likeprotein Complement C1q tumor necrosis factor- CRA_a FLRT1 LACTB,mitochondrial related protein 5 Plasminogen-related protein BNucleobindin-2 Galectin-3 Opticin Probable palmitoyltransferase ZDHHC16Phospholipase A2 Pancreatic prohormone Pre-small/secreted glycoproteinAngiopoietin-related protein 1 Proenkephalin-B Pregnancy-specificbeta-1-glycoprotein 6 Pentraxin-related protein PTX3 UPF0510 proteinC19orf63 Peptidoglycan recognition protein I-beta Dickkopf-relatedprotein 3 Carboxylesterase 8 Scavenger receptor cysteine-rich type 1Immunoglobulin superfamily containing Dehydrogenase/reductase SDR familyThioredoxin-related transmembrane protein M160 leucine-rich repeatprotein 2 member 11 protein 4 ER degradation-enhancing alpha- V-set andimmunoglobulin domain- Regenerating islet-derived protein 3 Majorfacilitator superfamily domain- mannosidase-like 2 containing protein 2gamma containing protein 2 Beta-galactosidase-1-like protein 2 PeptideYY RING finger protein 43 Kallikrein-12 Interleukin-17 receptor ERetinol-binding protein 3 Semenogelin-2 Brevican core proteinInterleukin-20 Atherin Mucin-15 Porimin Interleukin-25 Translocationprotein SEC63 homolog Bone sialoprotein 2 Torsin-1A PDZdomain-containing protein 11 Transforming growth factor beta-3Lymphotactin C-C motif chemokine 23 Relaxin-3 Protein Wnt-10bGrowth-regulated alpha protein Testican-3 Retinoid-inducible serinecarboxypeptidase Renalase R-spondin-2 Basic salivary proline-richprotein 4 Short palate, lung and nasal epithelium Proprotein convertasesubtilisin/kexin Transmembrane and coiled-coil domain- Tumor necrosisfactor receptor carcinoma-associated protein 2 type 4 containing protein3 superfamily member 18 WAP four-disulfide core domain protein 5Carboxypeptidase A4 VEGF co-regulated chemokine 1 Brother of CDOPlatelet-derived growth factor C Olfactomedin-4 ADM2Beta-1,4-galactosyltransferase 4 Disintegrin and metalloproteinasedomain- Insulin-like growth factor-binding protein Hydroxysteroid11-beta-dehydrogenase Dehydrogenase/reductase SDR family containingprotein 33 complex acid labile chain 1-like protein member 9 BSDdomain-containing protein 1 Amelogenin, Y isoform Delta-like protein 1Eppin Cell adhesion molecule 3 Arylsulfatase F Ephrin-A1 OtoancorinCDC45-related protein Choriogonadotropin subunit beta variant 2Fibroblast growth factor receptor-like 1 Tenascin-R ChondrolectinBeta-defensin 104 GDNF family receptor alpha-3 Growth factorDiacylglycerol O-acyltransferase 2 Beta-defensin 105 Platelet receptorGi24 Protein TSPEAR 3-keto-steroid reductase Beta-defensin 107Progonadoliberin-2 Hephaestin Interleukin-17 receptor C Protein WFDC11Kallikrein-7 Butyrophilin-like protein 3 Interleukin-17 receptor D WAPfour-disulfide core domain protein 6 Apolipoprotein F Butyrophilin-likeprotein 9 Integrator complex subunit 1 Epigen Protein CASC4 Lamininsubunit gamma-2 Junctional adhesion molecule-like Protein FAM19A5VIP36-like protein Protein LMBR1L E3 ubiquitin-protein ligase LNXClaudin-6 Magnesium transporter protein 1 Mucin-21 Leucine-rich repeattransmembrane Carcinoembryonic antigen-related cell Amiloride-sensitiveamine oxidase Endoplasmic reticulum mannosyl- neuronal protein 3adhesion molecule 19 [copper-containing] oligosaccharide1,2-alpha-mannosidase Methionine adenosyltransferase 2 A disintegrin andmetalloproteinase with DNA damage-regulated autophagy Pancreaticsecretory granule membrane subunit beta thrombospondin motifs 1modulator protein 2 major glycoprotein GP2 Podocalyxin-like protein 2Protein COQ10 A, mitochondrial Transmembrane protein C17orf87Semaphorin-4B Prominin-2 Uncharacterized protein C19orf41 Complementfactor H-related protein 5 Semaphorin-5B Plexin domain-containingprotein 2 Uncharacterized protein C21orf63 FK506-binding protein 7Epsilon-sarcoglycan Roundabout homolog 4 Protein delta homolog 2 Serineincorporator 1 Guanylate-binding protein 5 Lactosylceramide alpha-2,3-Cocaine- and amphetamine-regulated Transmembrane and ubiquitin-likeEctonucleoside triphosphate sialyltransferase transcript proteindomain-containing protein 1 diphosphohydrolase 6 SID1 transmembranefamily member 2 Lipoma HMGIC fusion partner-like 1 protein ProteinERGIC-53-like Serpin B3 Sushi domain-containing protein 1 Leucine-richrepeat-containing protein 18 Toll-like receptor 10 Protein RMD5 homologB Serine/threonine-protein kinase TAO2 Leucine-rich repeat-containingprotein 25 Toll-like receptor 8 Scavenger receptor class A member 5Transmembrane protease, serine 2 Leucine-rich repeat-containing protein3B Selenoprotein T Semaphorin-6B UDP-glucuronic acid decarboxylase 1Leucine-rich repeat-containing protein 3 Sialic acid-binding Ig-likelectin 11 Transmembrane protein 108 Uncharacterized protein C10orf58Ly6/PLAUR domain-containing protein 4 Sorting nexin-24 Sushidomain-containing protein 3 Thioredoxin-related transmembrane Vitamin Kepoxide reductase complex Complement C1q tumor necrosis factor-Latent-transforming growth factor beta- protein 2 subunit 1 relatedprotein 1 binding protein 2 CMP-N-acetylneuraminate-beta- A disintegrinand metalloproteinase with Putative uncharacterized protein Putativeuncharacterized protein galactosamide-alpha-2,3-sialyltransferasethrombospondin motifs 20 UNQ6494/PRO21346 UNQ6190/PRO20217 Putativeuncharacterized protein Putative uncharacterized protein Secreted andtransmembrane 1 precusor Secreted and transmembrane 1 precusorENSP00000380674 ENSP00000381830 variant variant Transmembrane protein119 Cat eye syndrome critical region protein 1 C-type lectin domainfamily 18 member A Collagen alpha-1(XX) chain Transmembrane protein 98Testis-expressed protein 101 Cysteine-rich secretory protein 3 Netrinreceptor UNC5D Pre-B lymphocyte protein 3 Xylosyltransferase 2Complement C4-A Mucin-13 Putative uncharacterized protein C14orf144Protein FAM20A Putative uncharacterized protein ATP-dependentmetalloprotease YME1L1 PRO2829 Membrane-bound transcription factorsite-1 Transmembrane and immunoglobulin Calcium-activated chloridechannel Proprotein convertase subtilisin/kexin proteasedomain-containing protein 1 regulator 2 type 5 Ficolin(Collagen/fibrinogen domain Putative killer cell immunoglobulin-likeNeuroblastoma suppressor of containing) 3 (Hakata antigen) (NL3)receptor-like protein KIR3DX1 (Leukocyte tumorigenicity 1 (Ficolin(Collagen/fibrinogen domain receptor cluster member 12) containing) 3(Hakata antigen), isoform CRA_b)

The therapeutic proteins provided herein should not be considered to beexclusive. Rather, as is apparent from the disclosure provided herein,the methods of the invention are applicable to any protein whereinattachment of a water soluble polymer is desired according to theinvention. For example, therapeutic proteins are described in US2007/0026485, incorporated herein by reference in its entirety.

Blood Coagulation Proteins

In one aspect, the starting material of the present invention is a bloodcoagulation protein, which can be derived from human plasma, or producedby recombinant engineering techniques, as described in U.S. Pat. No.4,757,006; U.S. Pat. No. 5,733,873; U.S. Pat. No. 5,198,349; U.S. Pat.No. 5,250,421; U.S. Pat. No. 5,919,766; and EP 306 968.

Therapeutic polypeptides such as blood coagulation proteins includingFactor IX (FIX), Factor VIII (FVIII), Factor VIIa (FVIIa), VonWillebrand Factor (VWF), Factor FV (FV), Factor X (FX), Factor XI (FXI),Factor XII (FXII), thrombin (FII), protein C, protein S, tPA, PAI-1,tissue factor (TF) and ADAMTS 13 protease are rapidly degraded byproteolytic enzymes and neutralized by antibodies. This reduces theirhalf-life and circulation time, thereby limiting their therapeuticeffectiveness. Relatively high doses and frequent administration arenecessary to reach and sustain the desired therapeutic or prophylacticeffect of these coagulation proteins. As a consequence, adequate doseregulation is difficult to obtain and the need of frequent intravenousadministrations imposes restrictions on the patient's way of living.

As described herein, blood coagulation proteins including, but notlimited to, Factor IX (FIX), Factor VIII (FVIII), Factor VIIa (FVIIa),Von Willebrand Factor (VWF), Factor FV (FV), Factor X (FX), Factor XI,Factor XII (FXII), thrombin (FII), protein C, protein S, tPA, PAI-1,tissue factor (TF) and ADAMTS 13 protease are contemplated by theinvention. As used herein, the term “blood coagulation protein” refersto any Factor IX (FIX), Factor VIII (FVIII), Factor VIIa (FVIIa), VonWillebrand Factor (VWF), Factor FV (FV), Factor X (FX), Factor XII(FXII), thrombin (FII), protein C, protein S, tPA, PAI-1, tissue factor(TF) and ADAMTS 13 protease which exhibits biological activity that isassociated with that particular native blood coagulation protein.

The blood coagulation cascade is divided into three distinct segments:the intrinsic, extrinsic, and common pathways (Schenone et al., CurrOpin Hematol. 2004; 11:272-7). The cascade involves a series of serineprotease enzymes (zymogens) and protein cofactors. When required, aninactive zymogen precursor is converted into the active form, whichconsequently converts the next enzyme in the cascade.

The intrinsic pathway requires the clotting factors VIII, IX, X, XI, andXII. Initiation of the intrinsic pathway occurs when prekallikrein,high-molecular-weight kininogen, factor XI (FXI) and factor XII (FXII)are exposed to a negatively charged surface. Also required are calciumions and phospholipids secreted from platelets.

The extrinsic pathway is initiated when the vascular lumen of bloodvessels is damaged. The membrane glycoprotein tissue factor is exposedand then binds to circulating factor VII (FVII) and to small preexistingamounts of its activated form FVIIa. This binding facilitates fullconversion of FVII to FVIIa and subsequently, in the presence of calciumand phospholipids, the conversion of factor IX (FIX) to factor IXa(FIXa) and factor X (FX) to factor Xa (FXa). The association of FVIIawith tissue factor enhances the proteolytic activity by bringing thebinding sites of FVII for the substrate (FIX and FX) into closerproximity and by inducing a conformational change, which enhances theenzymatic activity of FVIIa.

The activation of FX is the common point of the two pathways. Along withphospholipid and calcium, factors Va (FVa) and Xa convert prothrombin tothrombin (prothrombinase complex), which then cleaves fibrinogen to formfibrin monomers. The monomers polymerize to form fibrin strands. FactorXIIIa (FXIIIa) covalently bonds these strands to one another to form arigid mesh.

Conversion of FVII to FVIIa is also catalyzed by a number of proteases,including thrombin, FIXa, FXa, factor XIa (FXIa), and factor XIIa(FXIIa). For inhibition of the early phase of the cascade, tissue factorpathway inhibitor targets FVIIa/tissue factor/FXa product complex.

Factor VIIa

FVII (also known as stable factor or proconvertin) is a vitaminK-dependent serine protease glycoprotein with a pivotal role inhemostasis and coagulation (Eigenbrot, Curr Protein Pept Sci. 2002;3:287-99).

FVII is synthesized in the liver and secreted as a single-chainglycoprotein of 48 kD. FVII shares with all vitamin K-dependent serineprotease glycoproteins a similar protein domain structure consisting ofan amino-terminal gamma-carboxyglutamic acid (Gla) domain with 9-12residues responsible for the interaction of the protein with lipidmembranes, a carboxy-terminal serine protease domain (catalytic domain),and two epidermal growth factor-like domains containing a calcium ionbinding site that mediates interaction with tissue factor.Gamma-glutamyl carboxylase catalyzes carboxylation of Gla residues inthe amino-terminal portion of the molecule. The carboxylase is dependenton a reduced form of vitamin K for its action, which is oxidized to theepoxide form. Vitamin K epoxide reductase is required to convert theepoxide form of vitamin K back to the reduced form.

The major proportion of FVII circulates in plasma in zymogen form, andactivation of this form results in cleavage of the peptide bond betweenarginine 152 and isoleucine 153. The resulting activated FVIIa consistsof a NH2-derived light chain (20 kD) and a COOH terminal-derived heavychain (30 kD) linked via a single disulfide bond (Cys 135 to Cys 262).The light chain contains the membrane-binding Gla domain, while theheavy chain contains the catalytic domain.

The plasma concentration of FVII determined by genetic and environmentalfactors is about 0.5 mg/mL (Pinotti et al., Blood. 2000; 95:3423-8).Different FVII genotypes can result in several-fold differences in meanFVII levels. Plasma FVII levels are elevated during pregnancy in healthyfemales and also increase with age and are higher in females and inpersons with hypertriglyceridemia. FVII has the shortest half-life ofall procoagulant factors (3-6 h). The mean plasma concentration of FVIIais 3.6 ng/mL in healthy individuals and the circulating half-life ofFVIIa is relatively long (2.5 h) compared with other coagulationfactors.

Hereditary FVII deficiency is a rare autosomal recessive bleedingdisorder with a prevalence estimated to be 1 case per 500,000 persons inthe general population (Acharya et al., J Thromb Haemost. 2004;2248-56). Acquired FVII deficiency from inhibitors is also very rare.Cases have also been reported with the deficiency occurring inassociation with drugs such as cephalosporins, penicillins, and oralanticoagulants. Furthermore, acquired FVII deficiency has been reportedto occur spontaneously or with other conditions, such as myeloma,sepsis, aplastic anemia, with interleukin-2 and antithymocyte globulintherapy.

Reference polynucleotide and polypeptide sequences include, e.g.,GenBank Accession Nos. J02933 for the genomic sequence, M13232 for thecDNA (Hagen et al. PNAS 1986; 83: 2412-6), and P08709 for thepolypeptide sequence (references incorporated herein in theirentireties). A variety of polymorphisms of FVII have been described, forexample see Sabater-Lleal et al. (Hum Genet. 2006; 118:741-51)(reference incorporated herein in its entirety).

Factor IX

FIX is a vitamin K-dependent plasma protein that participates in theintrinsic pathway of blood coagulation by converting FX to its activeform in the presence of calcium ions, phospholipids and FVIIIa. Thepredominant catalytic capability of FIX is as a serine protease withspecificity for a particular arginine-isoleucine bond within FX.Activation of FIX occurs by FXIa which causes excision of the activationpeptide from FIX to produce an activated FIX molecule comprising twochains held by one or more disulphide bonds. Defects in FIX are thecause of recessive X-linked hemophilia B.

Hemophilia A and B are inherited diseases characterized by deficienciesin FVIII and FIX polypeptides, respectively. The underlying cause of thedeficiencies is frequently the result of mutations in FVIII and FIXgenes, both of which are located on the X chromosome. Traditionaltherapy for hemophilias often involves intravenous administration ofpooled plasma or semi-purified coagulation proteins from normalindividuals. These preparations can be contaminated by pathogenic agentsor viruses, such as infectious prions, HIV, parvovirus, hepatitis A, andhepatitis C. Hence, there is an urgent need for therapeutic agents thatdo not require the use of human serum.

The level of the decrease in FIX activity is directly proportional tothe severity of hemophilia B. The current treatment of hemophilia Bconsists of the replacement of the missing protein by plasma-derived orrecombinant FIX (so-called FIX substitution or replacement treatment ortherapy).

Polynucleotide and polypeptide sequences of FIX can be found for examplein the UniProtKB/Swiss-Prot Accession No. P00740, U.S. Pat. No.6,531,298 and in FIG. 1 (SEQ ID NO: 1).

Factor VIII

Coagulation factor VIII (FVIII) circulates in plasma at a very lowconcentration and is bound non-covalently to Von Willebrand factor(VWF). During hemostasis, FVIII is separated from VWF and acts as acofactor for activated factor IX (FIXa)-mediated FX activation byenhancing the rate of activation in the presence of calcium andphospholipids or cellular membranes.

FVIII is synthesized as a single-chain precursor of approximately270-330 kD with the domain structure A1-A2-B-A3-C1-C2. When purifiedfrom plasma (e.g., “plasma-derived” or “plasmatic”), FVIII is composedof a heavy chain (A1-A2-B) and a light chain (A3-C1-C2). The molecularmass of the light chain is 80 kD whereas, due to proteolysis within theB domain, the heavy chain is in the range of 90-220 kD.

FVIII is also synthesized as a recombinant protein for therapeutic usein bleeding disorders. Various in vitro assays have been devised todetermine the potential efficacy of recombinant FVIII (rFVIII) as atherapeutic medicine. These assays mimic the in vivo effects ofendogenous FVIII. In vitro thrombin treatment of FVIII results in arapid increase and subsequent decrease in its procoagulant activity, asmeasured by in vitro assays. This activation and inactivation coincideswith specific limited proteolysis both in the heavy and the lightchains, which alter the availability of different binding epitopes inFVIII, e.g. allowing FVIII to dissociate from VWF and bind to aphospholipid surface or altering the binding ability to certainmonoclonal antibodies.

The lack or dysfunction of FVIII is associated with the most frequentbleeding disorder, hemophilia A. The treatment of choice for themanagement of hemophilia A is replacement therapy with plasma derived orrFVIII concentrates. Patients with severe hemophilia A with FVIII levelsbelow 1%, are generally on prophylactic therapy with the aim of keepingFVIII above 1% between doses. Taking into account the average half-livesof the various FVIII products in the circulation, this result canusually be achieved by giving FVIII two to three times a week.

Reference polynucleotide and polypeptide sequences include, e.g.,UniProtKB/Swiss-Prot P00451 (FA8_HUMAN); Gitschier J et al.,Characterization of the human Factor VIII gene, Nature, 312(5992):326-30 (1984); Vehar G H et al., Structure of human Factor VIII, Nature,312(5992):337-42 (1984); Thompson A R. Structure and Function of theFactor VIII gene and protein, Semin Thromb Hemost, 2003:29; 11-29(2002).

Von Willebrand Factor

Von Willebrand factor (VWF) is a glycoprotein circulating in plasma as aseries of multimers ranging in size from about 500 to 20,000 kD.Multimeric forms of VWF are composed of 250 kD polypeptide subunitslinked together by disulfide bonds. VWF mediates initial plateletadhesion to the sub-endothelium of the damaged vessel wall. Only thelarger multimers exhibit hemostatic activity. It is assumed thatendothelial cells secrete large polymeric forms of VWF and those formsof VWF which have a low molecular weight (low molecular weight VWF)arise from proteolytic cleavage. The multimers having large molecularmasses are stored in the Weibel-Pallade bodies of endothelial cells andliberated upon stimulation.

VWF is synthesized by endothelial cells and megakaryocytes as prepro-VWFthat consists to a large extent of repeated domains. Upon cleavage ofthe signal peptide, pro-VWF dimerizes through disulfide linkages at itsC-terminal region. The dimers serve as protomers for multimerization,which is governed by disulfide linkages between the free end termini.The assembly to multimers is followed by the proteolytic removal of thepropeptide sequence (Leyte et al., Biochem. J. 274 (1991), 257-261).

The primary translation product predicted from the cloned cDNA of VWF isa 2813-residue precursor polypeptide (prepro-VWF). The prepro-VWFconsists of a 22 amino acid signal peptide and a 741 amino acidpropeptide, with the mature VWF comprising 2050 amino acids (Ruggeri Z.A., and Ware, J., FASEB J., 308-316 (1993).

Defects in VWF are causal to Von Willebrand disease (VWD), which ischaracterized by a more or less pronounced bleeding phenotype. VWD type3 is the most severe form, in which VWF is completely missing, and VWDtype 1 relates to a quantitative loss of VWF and its phenotype can bevery mild. VWD type 2 relates to qualitative defects of VWF and can beas severe as VWD type 3. VWD type 2 has many sub forms, some beingassociated with the loss or the decrease of high molecular weightmultimers. Von Willebrand disease type 2a (VWD-2A) is characterized by aloss of both intermediate and large multimers. VWD-2B is characterizedby a loss of highest-molecular-weight multimers. Other diseases anddisorders related to VWF are known in the art.

The polynucleotide and amino acid sequences of prepro-VWF are availableat GenBank Accession Nos. NM_000552 and NP_000543, respectively.

Other blood coagulation proteins according to the present invention aredescribed in the art, e.g. Mann K G, Thromb Haemost, 1999; 82:165-74.

A. Polypeptides

In one aspect, the starting material of the present invention is aprotein or polypeptide. As described herein, the term therapeuticprotein refers to any therapeutic protein molecule which exhibitsbiological activity that is associated with the therapeutic protein. Inone embodiment of the invention, the therapeutic protein molecule is afull-length protein.

Therapeutic protein molecules contemplated include full-length proteins,precursors of full length proteins, biologically active subunits orfragments of full length proteins, as well as biologically activederivatives and variants of any of these forms of therapeutic proteins.Thus, therapeutic protein include those that (1) have an amino acidsequence that has greater than about 60%, about 65%, about 70%, about75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%,about 94%, about 95%, about 96%, about 97%, about 98% or about 99% orgreater amino acid sequence identity, over a region of at least about25, about 50, about 100, about 200, about 300, about 400, or more aminoacids, to a polypeptide encoded by a referenced nucleic acid or an aminoacid sequence described herein; and/or (2) specifically bind toantibodies, e.g., polyclonal or monoclonal antibodies, generated againstan immunogen comprising a referenced amino acid sequence as describedherein, an immunogenic fragment thereof, and/or a conservativelymodified variant thereof.

According to the present invention, the term “recombinant therapeuticprotein” includes any therapeutic protein obtained via recombinant DNAtechnology. In certain embodiments, the term encompasses proteins asdescribed herein.

As used herein, “endogenous therapeutic protein” includes a therapeuticprotein which originates from the mammal intended to receive treatment.The term also includes therapeutic protein transcribed from a transgeneor any other foreign DNA present in said mammal. As used herein,“exogenous therapeutic protein” includes a blood coagulation proteinwhich does not originate from the mammal intended to receive treatment.

As used herein, “plasma-derived blood coagulation protein” or“plasmatic” includes all forms of the protein found in blood obtainedfrom a mammal having the property participating in the coagulationpathway.

As used herein “biologically active derivative” or “biologically activevariant” includes any derivative or variant of a molecule havingsubstantially the same functional and/or biological properties of saidmolecule, such as binding properties, and/or the same structural basis,such as a peptidic backbone or a basic polymeric unit.

An “analog,” such as a “variant” or a “derivative,” is a compoundsubstantially similar in structure and having the same biologicalactivity, albeit in certain instances to a differing degree, to anaturally-occurring molecule. For example, a polypeptide variant refersto a polypeptide sharing substantially similar structure and having thesame biological activity as a reference polypeptide. Variants or analogsdiffer in the composition of their amino acid sequences compared to thenaturally-occurring polypeptide from which the analog is derived, basedon one or more mutations involving (i) deletion of one or more aminoacid residues at one or more termini of the polypeptide and/or one ormore internal regions of the naturally-occurring polypeptide sequence(e.g., fragments), (ii) insertion or addition of one or more amino acidsat one or more termini (typically an “addition” or “fusion”) of thepolypeptide and/or one or more internal regions (typically an“insertion”) of the naturally-occurring polypeptide sequence or (iii)substitution of one or more amino acids for other amino acids in thenaturally-occurring polypeptide sequence. By way of example, a“derivative” is a type of analog and refers to a polypeptide sharing thesame or substantially similar structure as a reference polypeptide thathas been modified, e.g., chemically.

A variant polypeptide is a type of analog polypeptide and includesinsertion variants, wherein one or more amino acid residues are added toa therapeutic protein amino acid sequence of the invention. Insertionsmay be located at either or both termini of the protein, and/or may bepositioned within internal regions of the therapeutic protein amino acidsequence. Insertion variants, with additional residues at either or bothtermini, include for example, fusion proteins and proteins includingamino acid tags or other amino acid labels. In one aspect, the bloodcoagulation protein molecule optionally contains an N-terminal Met,especially when the molecule is expressed recombinantly in a bacterialcell such as E. coli.

In deletion variants, one or more amino acid residues in a therapeuticprotein polypeptide as described herein are removed. Deletions can beeffected at one or both termini of the therapeutic protein polypeptide,and/or with removal of one or more residues within the therapeuticprotein amino acid sequence. Deletion variants, therefore, includefragments of a therapeutic protein polypeptide sequence.

In substitution variants, one or more amino acid residues of atherapeutic protein polypeptide are removed and replaced withalternative residues. In one aspect, the substitutions are conservativein nature and conservative substitutions of this type are well known inthe art. Alternatively, the invention embraces substitutions that arealso non-conservative. Exemplary conservative substitutions aredescribed in Lehninger, [Biochemistry, 2nd Edition; Worth Publishers,Inc., New York (1975), pp. 71-77] and are set out immediately below.

CONSERVATIVE SUBSTITUTIONS SIDE CHAIN CHARACTERISTIC AMINO ACIDNon-polar (hydrophobic): A. Aliphatic A L I V P B. Aromatic F W C.Sulfur-containing M D. Borderline G Uncharged-polar: A. Hydroxyl S T YB. Amides N Q C. Sulfhydryl C D. Borderline G Positively charged (basic)K R H Negatively charged (acidic) D E

Alternatively, exemplary conservative substitutions are set outimmediately below.

CONSERVATIVE SUBSTITUTIONS II EXEMPLARY ORIGINAL RESIDUE SUBSTITUTIONAla (A) Val, Leu, Ile Arg (R) Lys, Gln, Asn Asn (N) Gln, His, Lys, ArgAsp (D) Glu Cys (C) Ser Gln (Q) Asn Glu (E) Asp His (H) Asn, Gln, Lys,Arg Ile (I) Leu, Val, Met, Ala, Phe, Leu (L) Ile, Val, Met, Ala, Phe Lys(K) Arg, Gln, Asn Met (M) Leu, Phe, Ile Phe (F) Leu, Val, Ile, Ala Pro(P) Gly Ser (S) Thr Thr (T) Ser Trp (W) Tyr Tyr (Y) Trp, Phe, Thr, SerVal (V) Ile, Leu, Met, Phe, Ala

B. Polynucleotides

Nucleic acids encoding a therapeutic protein of the invention include,for example and without limitation, genes, pre-mRNAs, mRNAs, cDNAs,polymorphic variants, alleles, synthetic and naturally-occurringmutants.

Polynucleotides encoding a therapeutic protein of the invention alsoinclude, without limitation, those that (1) specifically hybridize understringent hybridization conditions to a nucleic acid encoding areferenced amino acid sequence as described herein, and conservativelymodified variants thereof; (2) have a nucleic acid sequence that hasgreater than about 95%, about 96%, about 97%, about 98%, about 99%, orhigher nucleotide sequence identity, over a region of at least about 25,about 50, about 100, about 150, about 200, about 250, about 500, about1000, or more nucleotides (up to the full length sequence of 1218nucleotides of the mature protein), to a reference nucleic acid sequenceas described herein. Exemplary “stringent hybridization” conditionsinclude hybridization at 42° C. in 50% formamide, 5×SSC, 20 mM Na.PO4,pH 6.8; and washing in 1×SSC at 55° C. for 30 minutes. It is understoodthat variation in these exemplary conditions can be made based on thelength and GC nucleotide content of the sequences to be hybridized.Formulas standard in the art are appropriate for determining appropriatehybridization conditions. See Sambrook et al., Molecular Cloning: ALaboratory Manual (Second ed., Cold Spring Harbor Laboratory Press,1989) §§9.47-9.51.

A “naturally-occurring” polynucleotide or polypeptide sequence istypically derived from a mammal including, but not limited to, primate,e.g., human; rodent, e.g., rat, mouse, hamster; cow, pig, horse, sheep,or any mammal. The nucleic acids and proteins of the invention can berecombinant molecules (e.g., heterologous and encoding the wild typesequence or a variant thereof, or non-naturally occurring).

C. Production of Therapeutic Proteins

Production of a therapeutic protein includes any method known in the artfor (i) the production of recombinant DNA by genetic engineering, (ii)introducing recombinant DNA into prokaryotic or eukaryotic cells by, forexample and without limitation, transfection, electroporation ormicroinjection, (iii) cultivating said transformed cells, (iv)expressing therapeutic protein, e.g. constitutively or upon induction,and (v) isolating said blood coagulation protein, e.g. from the culturemedium or by harvesting the transformed cells, in order to obtainpurified therapeutic protein.

In other aspects, the therapeutic protein is produced by expression in asuitable prokaryotic or eukaryotic host system characterized byproducing a pharmacologically acceptable blood coagulation proteinmolecule. Examples of eukaryotic cells are mammalian cells, such as CHO,COS, HEK 293, BHK, SK-Hep, and HepG2.

A wide variety of vectors are used for the preparation of thetherapeutic protein and are selected from eukaryotic and prokaryoticexpression vectors. Examples of vectors for prokaryotic expressioninclude plasmids such as, and without limitation, pRSET, pET, and pBAD,wherein the promoters used in prokaryotic expression vectors include oneor more of, and without limitation, lac, trc, trp, recA, or araBAD.Examples of vectors for eukaryotic expression include: (i) forexpression in yeast, vectors such as, and without limitation, pAO, pPIC,pYES, or pMET, using promoters such as, and without limitation, AOX1,GAP, GAL1, or AUG1; (ii) for expression in insect cells, vectors such asand without limitation, pMT, pAc5, pIB, pMIB, or pBAC, using promoterssuch as and without limitation PH, p10, MT, Ac5, OpIE2, gp64, or polh,and (iii) for expression in mammalian cells, vectors such as and withoutlimitation pSVL, pCMV, pRc/RSV, pcDNA3, or pBPV, and vectors derivedfrom, in one aspect, viral systems such as and without limitationvaccinia virus, adeno-associated viruses, herpes viruses, orretroviruses, using promoters such as and without limitation CMV, SV40,EF-1, UbC, RSV, ADV, BPV, and β-actin.

D. Administration

In one embodiment a conjugated therapeutic protein of the presentinvention may be administered by injection, such as intravenous,intramuscular, or intraperitoneal injection.

To administer compositions comprising a conjugated therapeutic proteinof the present invention to human or test animals, in one aspect, thecompositions comprise one or more pharmaceutically acceptable carriers.The terms “pharmaceutically” or “pharmacologically acceptable” refer tomolecular entities and compositions that are stable, inhibit proteindegradation such as aggregation and cleavage products, and in additiondo not produce allergic, or other adverse reactions when administeredusing routes well-known in the art, as described below.“Pharmaceutically acceptable carriers” include any and all clinicallyuseful solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents and the like,including those agents disclosed above.

As used herein, “effective amount” includes a dose suitable for treatinga disease or disorder or ameliorating a symptom of a disease ordisorder. In one embodiment, “effective amount” includes a dose suitablefor treating a mammal having a bleeding disorder as described herein.

The compositions may be administered orally, topically, transdermally,parenterally, by inhalation spray, vaginally, rectally, or byintracranial injection. The term parenteral as used herein includessubcutaneous injections, intravenous, intramuscular, intracisternalinjection, or infusion techniques. Administration by intravenous,intradermal, intramuscular, intramammary, intraperitoneal, intrathecal,retrobulbar, intrapulmonary injection and or surgical implantation at aparticular site is contemplated as well. Generally, compositions areessentially free of pyrogens, as well as other impurities that could beharmful to the recipient.

Single or multiple administrations of the compositions can be carriedout with the dose levels and pattern being selected by the treatingphysician. For the prevention or treatment of disease, the appropriatedosage will depend on the type of disease to be treated, as describedabove, the severity and course of the disease, whether drug isadministered for preventive or therapeutic purposes, previous therapy,the patient's clinical history and response to the drug, and thediscretion of the attending physician.

The present invention also relates to a pharmaceutical compositioncomprising an effective amount of a conjugated therapeutic protein asdefined herein. The pharmaceutical composition may further comprise apharmaceutically acceptable carrier, diluent, salt, buffer, orexcipient. The pharmaceutical composition can be used for treating theabove-defined bleeding disorders. The pharmaceutical composition of theinvention may be a solution or a lyophilized product. Solutions of thepharmaceutical composition may be subjected to any suitablelyophilization process.

As an additional aspect, the invention includes kits which comprise acomposition of the invention packaged in a manner which facilitates itsuse for administration to subjects. In one embodiment, such a kitincludes a compound or composition described herein (e.g., a compositioncomprising a conjugated therapeutic protein), packaged in a containersuch as a sealed bottle or vessel, with a label affixed to the containeror included in the package that describes use of the compound orcomposition in practicing the method. In one embodiment, the kitcontains a first container having a composition comprising a conjugatedtherapeutic protein and a second container having a physiologicallyacceptable reconstitution solution for the composition in the firstcontainer. In one aspect, the compound or composition is packaged in aunit dosage form. The kit may further include a device suitable foradministering the composition according to a specific route ofadministration. Preferably, the kit contains a label that describes useof the therapeutic protein or peptide composition.

Water Soluble Polymers

In one aspect, a therapeutic protein derivative (i.e., a conjugatedtherapeutic protein) molecule provided is bound to a water-solublepolymer including, but not limited to, polyethylene glycol (PEG),branched PEG, polysialic acid (PSA), hydroxyalkyl starch (HAS),hydroxylethyl starch (HES), carbohydrate, polysaccharides, pullulane,chitosan, hyaluronic acid, chondroitin sulfate, dermatan sulfate,starch, dextran, carboxymethyl-dextran, polyalkylene oxide (PAO),polyalkylene glycol (PAG), polypropylene glycol (PPG) polyoxazoline,poly acryloylmorpholine, polyvinyl alcohol (PVA), polycarboxylate,polyvinylpyrrolidone, polyphosphazene, polyoxazoline,polyethylene-co-maleic acid anhydride, polystyrene-co-maleic acidanhydride, poly(1-hydroxymethylethylene hydroxymethylformal) (PHF),2-methacryloyloxy-2′-ethyltrimethylammoniumphosphate (MPC). In oneembodiment of the invention, the water soluble polymer is consisting ofsialic acid molecule having a molecular weight range of 350 to 120,000,500 to 100,000,1000 to 80,000, 1500 to 60,000, 2,000 to 45,000 Da, 3,000to 35,000 Da, and 5,000 to 25,000 Da. The coupling of the water solublepolymer can be carried out by direct coupling to the protein or vialinker molecules. One example of a chemical linker is MBPH(4-[4-N-Maleimidophenyl]butyric acid hydrazide) containing acarbohydrate-selective hydrazide and a sulfhydryl-reactive maleimidegroup (Chamow et al., J Biol Chem 1992; 267:15916-22). Other exemplaryand preferred linkers are described below.

In one embodiment, the derivative retains the full functional activityof native therapeutic protein products, and provides an extendedhalf-life in vivo, as compared to native therapeutic protein products.In another embodiment, the derivative retains at least 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, 100, 110, 120, 130, 140, or 150 percent (%) biologicalactivity relative to native blood coagulation protein. In a relatedaspect, the biological activities of the derivative and native bloodcoagulation protein are determined by the ratios of chromogenic activityto blood coagulation factor antigen value (blood coagulation factor:Chr:blood coagulation factor:Ag). In still another embodiment of theinvention, the half-life of the construct is decreased or increased 0.5,0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3, 4, 5, 6, 7, 8,9, or 10-fold relative to the in vivo half-life of native therapeuticprotein.

A. Sialic Acid and PSA

PSAs consist of polymers (generally homopolymers) of N-acetylneuraminicacid. The secondary amino group normally bears an acetyl group, but itmay instead bear a glycolyl group. Possible substituents on the hydroxylgroups include acetyl, lactyl, ethyl, sulfate, and phosphate groups.

Structure of Sialic Acid (N-Acetylneuraminic Acid)

PSAs and mPSAs generally comprise linear polymers consisting essentiallyof N-acetylneuraminic acid moieties linked by 2,8- or 2,9-glycosidiclinkages or combinations of these (e.g. alternating 2,8- and2,9-linkages). In particularly preferred PSAs and mPSAs, the glycosidiclinkages are α-2,8. Such PSAs and mPSAs are conveniently derived fromcolominic acids, and are referred to herein as “CAs” and “mCAs”. TypicalPSAs and mPSAs comprise at least 2, preferably at least 5, morepreferably at least 10 and most preferably at least 20N-acetylneuraminic acid moieties. Thus, they may comprise from 2 to 300N-acetylneuraminic acid moieties, preferably from 5 to 200N-acetylneuraminic acid moieties, or most preferably from 10 to 100N-acetylneuraminic acid moieties. PSAs and CAs preferably areessentially free of sugar moieties other than N-acetylneuraminic acid.Thus PSAs and CAs preferably comprise at least 90%, more preferably atleast 95% and most preferably at least 98% N-acetylneuraminic acidmoieties.

Where PSAs and CAs comprise moieties other than N-acetylneuraminic acid(as, for example in mPSAS and mCAs) these are preferably located at oneor both of the ends of the polymer chain. Such “other” moieties may, forexample, be moieties derived from terminal N-acetylneuraminic acidmoieties by oxidation or reduction.

For example, WO-A-0187922 describes such mPSAs and mCAs in which thenon-reducing terminal N-acetylneuraminic acid unit is converted to analdehyde group by reaction with sodium periodate. Additionally, WO2005/016974 describes such mPSAs and mCAs in which the reducing terminalN-acetylneuraminic acid unit is subjected to reduction to reductivelyopen the ring at the reducing terminal N-acetylneuraminic acid unit,whereby a vicinal diol group is formed, followed by oxidation to convertthe vicinal diol group to an aldehyde group.

Sialic acid rich glycoproteins bind selectin in humans and otherorganisms. They play an important role in human influenza infections.E.g., sialic acid can hide mannose antigens on the surface of host cellsor bacteria from mannose-binding lectin. This prevents activation ofcomplement. Sialic acids also hide the penultimate galactose residuethus preventing rapid clearance of the glycoprotein by the galactosereceptor on the hepatic parenchymal cells.

Structure of Colominic Acid (Homopolymer of N-Acetylneuraminic Acid)

Colominic acids (a sub-class of PSAs) are homopolymers ofN-acetylneuraminic acid (NANA) with a (2→8) ketosidic linkage, and areproduced, inter alia, by particular strains of Escherichia colipossessing K1 antigen. Colominic acids have many physiologicalfunctions. They are important as a raw material for drugs and cosmetics.

Comparative studies in vivo with polysialylated and unmodifiedasparaginase revealed that polysialylation increased the half-life ofthe enzyme (Fernandes and Gregoriadis, Biochimica Biophysica Acta 1341:26-34, 1997).

As used herein, “sialic acid moieties” includes sialic acid monomers orpolymers (“polysaccharides”) which are soluble in an aqueous solution orsuspension and have little or no negative impact, such as side effects,to mammals upon administration of the PSA-blood coagulation proteinconjugate in a pharmaceutically effective amount. The polymers arecharacterized, in one aspect, as having 1, 2, 3, 4, 5, 10, 20, 30, 40,50, 60, 70, 80, 90, 100, 200, 300, 400, or 500 sialic acid units. Incertain aspects, different sialic acid units are combined in a chain.

In one embodiment of the invention, the sialic acid portion of thepolysaccharide compound is highly hydrophilic, and in another embodimentthe entire compound is highly hydrophilic. Hydrophilicity is conferredprimarily by the pendant carboxyl groups of the sialic acid units, aswell as the hydroxyl groups. The saccharide unit may contain otherfunctional groups, such as, amine, hydroxyl or sulphate groups, orcombinations thereof. These groups may be present on naturally-occurringsaccharide compounds, or introduced into derivative polysaccharidecompounds.

The naturally occurring polymer PSA is available as a polydispersepreparation showing a broad size distribution (e.g. Sigma C-5762) andhigh polydispersity (PD). Because the polysaccharides are usuallyproduced in bacteria carrying the inherent risk of copurifyingendotoxins, the purification of long sialic acid polymer chains mayraise the probability of increased endotoxin content. Short PSAmolecules with 1-4 sialic acid units can also be synthetically prepared(Kang S H et al., Chem Commun. 2000; 227-8; Ress D K and Linhardt R J,Current Organic Synthesis. 2004; 1:31-46), thus minimizing the risk ofhigh endotoxin levels. However PSA preparations with a narrow sizedistribution and low polydispersity, which are also endotoxin-free, cannow be manufactured. Polysaccharide compounds of particular use for theinvention are, in one aspect, those produced by bacteria. Some of thesenaturally-occurring polysaccharides are known as glycolipids. In oneembodiment, the polysaccharide compounds are substantially free ofterminal galactose units.

B. Polyethylene Glycol (PEG) and Pegylation

In certain aspects, therapeutic proteins are conjugated to a watersoluble polymer by any of a variety of chemical methods (Roberts J M etal., Advan Drug Delivery Rev 2002; 54:459-76). For example, in oneembodiment a therapeutic protein is modified by the conjugation of PEGto free amino groups of the protein using N-hydroxysuccinimide (NHS)esters. In another embodiment the water soluble polymer, for examplePEG, is coupled to free SH groups using maleimide chemistry or thecoupling of PEG hydrazides or PEG amines to carbohydrate moieties of thetherapeutic protein after prior oxidation.

The conjugation is in one aspect performed by direct coupling (orcoupling via linker systems) of the water soluble polymer to atherapeutic protein under formation of stable bonds. In additiondegradable, releasable or hydrolysable linker systems are used incertain aspects the present invention (Tsubery et al. J Biol Chem 2004;279:38118-24/Greenwald et al., J Med Chem 1999; 42:3657-67/Zhao et al.,Bioconj Chem 2006; 17:341-51/WO2006/138572A2/U.S. Pat. No.7,259,224B2/U.S. Pat. No. 7,060,259B2).

In one embodiment of the invention, a therapeutic protein is modifiedvia lysine residues by use of polyethylene glycol derivatives containingan active N-hydroxysuccinimide ester (NHS) such as succinimidylsuccinate, succinimidyl glutarate or succinimidyl propionate. Thesederivatives react with the lysine residues of the therapeutic proteinunder mild conditions by forming a stable amide bond. In one embodimentof the invention, the chain length of the PEG derivative is 5,000 Da.Other PEG derivatives with chain lengths of 500 to 2,000 Da, 2,000 to5,000 Da, greater than 5,000 up to 10,000 Da or greater than 10,000 upto 20,000 Da, or greater than 20,000 up to 150,000 Da are used invarious embodiments, including linear and branched structures.

Alternative methods for the PEGylation of amino groups are, withoutlimitation, the chemical conjugation with PEG carbonates by formingurethane bonds, or the reaction with aldehydes or ketones by reductiveamination forming secondary amide bonds.

In one embodiment of the present invention a therapeutic proteinmolecule is chemically modified using PEG derivatives that arecommercially available. These PEG derivatives in alternative aspectshave linear or branched structures. Examples of PEG-derivativescontaining NHS groups are listed below.

The following PEG derivatives are non-limiting examples of thosecommercially available from Nektar Therapeutics (Huntsville, Ala.; seewww.nektar.com/PEG reagent catalog; Nektar Advanced PEGylation, pricelist 2005-2006):

Structure of a Branched PEG-Derivative (Nektar Therapeutics)

This reagent with branched structure is described in more detail byKozlowski et al. (BioDrugs 2001; 5:419-29).

Other non-limiting examples of PEG derivatives are commerciallyavailable from NOF Corporation (Tokyo, Japan; see www.nof.co.jp/english:Catalogue 2005)

General Structure of Linear PEG-Derivatives (NOF Corp.)

Structures of Branched PEG-derivatives (NOF Corp.):2,3-Bis(methylpolyoxyethylene-oxy)-1-(1,5-dioxo-5-succinimidyloxy,pentyloxy)propane

2,3-Bis(methylpolyoxyethylene-oxy)-1-(succinimidylcarboxypentyloxy)propane

These propane derivatives show a glycerol backbone with a 1,2substitution pattern. In the present invention branched PEG derivativesbased on glycerol structures with 1,3 substitution or other branchedstructures described in US2003/0143596A1 are also contemplated.

PEG derivatives with degradable (for example, hydrolysable) linkers asdescribed by Tsubery et al. (J Biol Chem 2004; 279:38118-24) andShechter et al. (WO04089280A3) are also contemplated.

Surprisingly, the PEGylated therapeutic protein of this inventionexhibits functional activity, combined with an extended half-life invivo. In addition the PEGylated rFVIII, FVIIa, FIX, or other bloodcoagulation factor seems to be more resistant against thrombininactivation.

C. Hydroxyalkyl Starch (HAS) and Hydroxylethyl Starch (HES)

In various embodiments of the present invention, a therapeutic proteinmolecule is chemically modified using hydroxyalkyl starch (HAS) orhydroxylethyl starch (HES) or derivatives thereof.

HES is a derivative of naturally occurring amylopectin and is degradedby alpha-amylase in the body. HES is a substituted derivative of thecarbo-hydrate polymer amylopectin, which is present in corn starch at aconcentration of up to 95% by weight. HES exhibits advantageousbiological properties and is used as a blood volume replacement agentand in hemodilution therapy in the clinics (Sommermeyer et al., 1987,Krankenhauspharmazie, 8 (8), 271-278; and Weidler et al., 1991,Arzneim.-Forschung/Drug Res. g 419 494-498).

Amylopectin consists of glucose moieties, wherein in the main chainalpha-1,4-glycosidic bonds are present and at the branching sitesalpha-1,6-glycosidic bonds are found. The physical-chemical propertiesof this molecule are mainly determined by the type of glycosidic bonds.Due to the nicked alpha-1,4-glycosidic bond, helical structures withabout six glucose-monomers per turn are produced. The physico-chemicalas well as the biochemical properties of the polymer can be modified viasubstitution. The introduction of a hydroxyethyl group can be achievedvia alkaline hydroxyethylation. By adapting the reaction conditions itis possible to exploit the different reactivity of the respectivehydroxy group in the unsubstituted glucose monomer with respect to ahydroxyethylation. Owing to this fact, the skilled person is able toinfluence the substitution pattern to a limited extent.

HAS refers to a starch derivative which has been substituted by at leastone hydroxyalkyl group. Therefore, the term hydroxyalkyl starch is notlimited to compounds where the terminal carbohydrate moiety compriseshydroxyalkyl groups R1, R2, and/or R3, but also refers to compounds inwhich at least one hydroxy group present anywhere, either in theterminal carbohydrate moiety and/or in the remaining part of the starchmolecule, HAS′, is substituted by a hydroxyalkyl group R1, R2, or R3.

The alkyl group may be a linear or branched alkyl group which may besuitably substituted. Preferably, the hydroxyalkyl group contains 1 to10 carbon atoms, more preferably from 1 to 6 carbon atoms, morepreferably from 1 to 4 carbon atoms, and even more preferably 2-4 carbonatoms. “Hydroxyalkyl starch” therefore preferably comprises hydroxyethylstarch, hydroxypropyl starch and hydroxybutyl starch, whereinhydroxyethyl starch and hydroxypropyl starch are particularly preferred.

Hydroxyalkyl starch comprising two or more different hydroxyalkyl groupsis also comprised in the present invention. The at least onehydroxyalkyl group comprised in HAS may contain two or more hydroxygroups. According to one embodiment, the at least one hydroxyalkyl groupcomprised HAS contains one hydroxy group.

The term HAS also includes derivatives wherein the alkyl group is mono-or polysubstituted. In one embodiment, the alkyl group is substitutedwith a halogen, especially fluorine, or with an aryl group, providedthat the HAS remains soluble in water. Furthermore, the terminal hydroxygroup a of hydroxyalkyl group may be esterified or etherified. HASderivatives are described in WO/2004/024776, which is incorporated byreference in its entirety.

D. Methods of Attachment

A therapeutic protein may be covalently linked to the polysaccharidecompounds by any of various techniques known to those of skill in theart. In various aspects of the invention, sialic acid moieties are boundto a therapeutic protein, e.g., FIX, FVIII, FVIIa or VWF, for example bythe method described in U.S. Pat. No. 4,356,170, which is hereinincorporated by reference.

Other techniques for coupling PSA to polypeptides are also known andcontemplated by the invention. For example, US Publication No.2007/0282096 describes conjugating an amine or hydrazide derivative of,e.g., PSA, to proteins. In addition, US Publication No. 2007/0191597describes PSA derivatives containing an aldehyde group for reaction withsubstrates (e.g., proteins) at the reducing end. These references areincorporated by reference in their entireties.

Various methods are disclosed at column 7, line 15, through column 8,line 5 of U.S. Pat. No. 5,846,951 (incorporated by reference in itsentirety). Exemplary techniques include linkage through a peptide bondbetween a carboxyl group on one of either the blood coagulation proteinor polysaccharide and an amine group of the blood coagulation protein orpolysaccharide, or an ester linkage between a carboxyl group of theblood coagulation protein or polysaccharide and a hydroxyl group of thetherapeutic protein or polysaccharide. Another linkage by which thetherapeutic protein is covalently bonded to the polysaccharide compoundis via a Schiff base, between a free amino group on the bloodcoagulation protein being reacted with an aldehyde group formed at thenon-reducing end of the polysaccharide by periodate oxidation (JenningsH J and Lugowski C, J. Immunol. 1981; 127:1011-8; Fernandes A I andGregoriadis G, Biochim Biophys Acta. 1997; 1341; 26-34). The generatedSchiff base is in one aspect stabilized by specific reduction withNaCNBH₃ to form a secondary amine. An alternative approach is thegeneration of terminal free amino groups in the PSA by reductiveamination with NH₄Cl after prior oxidation. Bifunctional reagents can beused for linking two amino or two hydroxyl groups. For example, PSAcontaining an amino group is coupled to amino groups of the protein withreagents like BS3 (Bis(sulfosuccinimidyl)suberate/Pierce, Rockford,Ill.). In addition heterobifunctional cross linking reagents likeSulfo-EMCS(N-ε-Maleimidocaproyloxy)sulfosuccinimide ester/Pierce) isused for instance to link amine and thiol groups.

In another approach, a PSA hydrazide is prepared and coupled to thecarbohydrate moiety of the protein after prior oxidation and generationof aldehyde functions.

As described above, a free amine group of the therapeutic protein reactswith the 1-carboxyl group of the sialic acid residue to form a peptidylbond or an ester linkage is formed between the 1-carboxylic acid groupand a hydroxyl or other suitable active group on a blood coagulationprotein. Alternatively, a carboxyl group forms a peptide linkage withdeacetylated 5-amino group, or an aldehyde group of a molecule of atherapeutic protein forms a Schiff base with the N-deacetylated 5-aminogroup of a sialic acid residue.

Alternatively, the polysaccharide compound is associated in anon-covalent manner with a therapeutic protein. For example, thepolysaccharide compound and the pharmaceutically active compound are inone aspect linked via hydrophobic interactions. Other non-covalentassociations include electrostatic interactions, with oppositely chargedions attracting each other.

In various embodiments, the therapeutic protein is linked to orassociated with the polysaccharide compound in stoichiometric amounts(e.g., 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:7, 1:8, 1:9, or 1:10, etc.).In various embodiments, 1-6, 7-12 or 13-20 polysaccharides are linked tothe blood coagulation protein. In still other embodiments, 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or morepolysaccharides are linked to the blood coagulation protein.

In various embodiments, the therapeutic protein is modified to introduceglycosylation sites (i.e., sites other than the native glycosylationsites). Such modification may be accomplished using standard molecularbiological techniques known in the art. Moreover, the therapeuticprotein, prior to conjugation to a water soluble polymer via one or morecarbohydrate moieties, may be glycosylated in vivo or in vitro. Theseglycosylated sites can serve as targets for conjugation of the proteinswith water soluble polymers (US Patent Application No. 20090028822, USPatent Application No. 2009/0093399, US Patent Application No.2009/0081188, US Patent Application No. 2007/0254836, US PatentApplication No. 2006/0111279, and DeFrees S. et al., Glycobiology, 2006,16, 9, 833-43). For example, a protein that is not naturallyglycoslyated in vivo (e.g., a protein that is not a glycoprotein) may bemodified as described above.

E. Aminooxy Linkage

In one embodiment of the invention, the reaction of hydroxylamine orhydroxylamine derivatives with aldehydes (e.g., on a carbohydrate moietyfollowing oxidation by sodium periodate) to form an oxime group isapplied to the preparation of conjugates of blood coagulation protein.For example, a glycoprotein (e.g., a therapeutic protein according tothe present invention) is first oxidized with a oxidizing agent such assodium periodate (NaIO₄) (Rothfus J A et Smith E L., J Biol Chem 1963,238, 1402-10; and Van Lenten L and Ashwell G., J Biol Chem 1971, 246,1889-94). The periodate oxidation of glycoproteins is based on theclassical Malaprade reaction described in 1928, the oxidation of vicinaldiols with periodate to form an active aldehyde group (Malaprade L.,Analytical application, Bull Soc Chim France, 1928, 43, 683-96).Additional examples for such an oxidizing agent are lead tetraacetate(Pb(OAc)4), manganese acetate (MnO(Ac)3), cobalt acetate (Co(OAc)2),thallium acetate (TiOAc), cerium sulfate (Ce(SO4)2) (U.S. Pat. No.4,367,309) or potassium perruthenate (KRuO4) (Marko et al., J Am ChemSoc 1997, 119, 12661-2). By “oxidizing agent” a mild oxidizing compoundwhich is capable of oxidizing vicinal diols in carbohydrates, therebygenerating active aldehyde groups under physiological reactionconditions is meant.

The second step is the coupling of the polymer containing an aminooxygroup to the oxidized carbohydrate moiety to form an oxime linkage. Inone embodiment of the invention, this step can be carried out in thepresence of catalytic amounts of the nucleophilic catalyst aniline oraniline derivatives (Dirksen A et Dawson P E, Bioconjugate Chem. 2008;Zeng Y et al., Nature Methods 2009; 6:207-9). The aniline catalysisdramatically accelerates the oxime ligation allowing the use of very lowconcentrations of the reagents. In another embodiment of the inventionthe oxime linkage is stabilized by reduction with NaCNBH₃ to form analkoxyamine linkage (FIG. 2). Additional catalysts are described below.

Additional information on aminooxy technology can be found in thefollowing references, each of which is incorporated in their entireties:EP 1681303A1 (HASylated erythropoietin); WO 2005/014024 (conjugates of apolymer and a protein linked by an oxime linking group); WO96/40662(aminooxy-containing linker compounds and their application inconjugates); WO 2008/025856 (Modified proteins); Peri F et al.,Tetrahedron 1998, 54, 12269-78; Kubler-Kielb J et. Pozsgay V., J OrgChem 2005, 70, 6887-90; Lees A et al., Vaccine 2006, 24(6), 716-29; andHeredia K L et al., Macromolecules 2007, 40(14), 4772-9.

Numerous methods of coupling a water-soluble polymer to an aminooxylinker are contemplated by the present disclosure. For example, couplingof a linker to either the reducing or non-reducing end of awater-soluble polymer such as PSA is described herein. The coupling site(e.g., reducing end versus non-reducing end) is determined by one ormore conditions (e.g., time and temperature) of the coupling process aswell as the state (e.g., native versus oxidized) of the water-solublepolymer. In one embodiment, an oxidized water-soluble polymer such asPSA is coupled at it's non-reducing end to an aminooxy linker byperforming the coupling reaction at a reduced temperature (e.g., between2-8° C.). In another embodiment, a native (e.g., non-oxidized)water-soluble polymer such as PSA is coupled at it's reducing end to anaminooxy linker by performing the coupling reaction at a highertemperature (e.g., between 22-37° C.). The aforementioned embodimentsare described in more detail below and in the Examples.

As described herein, the reaction of oxidized PSA with a diaminooxylinker shows two reactions: a “quick reaction” of the aldehyde group atthe non-reducing end, and a “slow reaction” at the reducing end. Ifnative PSA (which is not oxidized and does not contain an activealdehyde group) is reacted with the reducing end at room temperature, aderivatized PSA can be observed. Thus, in various embodiments, in orderto minimize an unwanted side reaction at the reducing end of awater-soluble polymer such as PSA, the PSA-aminooxy linker reagentpreparation is performed at a temperature between 2-8° C.

In still another embodiment of the present disclosure, thederivatization of native PSA at the reducing end is provided. Asdescribed herein, native PSA (which is not oxidized by NaIO₄ and thusdoes not contain a free aldehyde group at its non-reducing end) isreacted with a diaminooxy linker at room temperature, a derivatizationof the PSA at its reducing end can be observed. This coupling occursthrough ring opening at the reducing end and subsequent oxime formation(the actual side reaction described above and the cause for the presenceof by-product in the aminooxy-PSA reagent). This reaction can beperformed with native PSA yielding in a degree of modification up toapproximately 70%.

As a main product the following structure was determined by 13C NMRspectroscopy

The reaction can be transferred to other carbohydrates like dextran andstarch or other polysaccharides containing reducing end groups. The useof a nucleophilic catalyst like m-toluidine or aniline is alsocontemplated. Thus, preparation of aminooxy-PSA reagents using nativePSA (i.e. without prior oxidation), which can then be used for chemicalmodification of therapeutic proteins, is provided herein.

Thus, in various embodiments of the present disclosure, methods areprovided wherein the conditions of coupling, (e.g., 2-8° C. incubationtemperature) a diaminooxy linker to a water soluble polymer such asoxidized PSA, favor the coupling to either the non-reducing end or, inone alternative, wherein the conditions of coupling (e.g., roomtemperature incubation) a diaminooxy linker to a water soluble polymersuch as native, non-oxidized PSA, favor the coupling to either thereducing end.

In various embodiments of the invention, the water soluble polymer whichis linked according to the aminooxy technology described herein to anoxidized carbohydrate moiety of a therapeutic protein (e.g., FVIII,FVIIa, or FIX) include, but are not limited to polyethylene glycol(PEG), branched PEG, polysialic acid (PSA), carbohydrate,polysaccharides, pullulane, chitosan, hyaluronic acid, chondroitinsulfate, dermatan sulfate, starch, dextran, carboxymethyl-dextran,polyalkylene oxide (PAO), polyalkylene glycol (PAG), polypropyleneglycol (PPG) polyoxazoline, poly acryloylmorpholine, polyvinyl alcohol(PVA), polycarboxylate, polyvinylpyrrolidone, polyphosphazene,polyoxazoline, polyethylene-co-maleic acid anhydride,polystyrene-co-maleic acid anhydride, poly(1-hydroxymethylethylenehydroxymethylformal) (PHF),2-methacryloyloxy-2′-ethyltrimethylammoniumphosphate (MPC).

Nucleophilic Catalysts

As described herein, the conjugation of water soluble polymers totherapeutic proteins can be catalyzed by aniline. Aniline stronglycatalyzes aqueous reactions of aldehydes and ketones with amines to formstable imines such as hydrazones and oximes. The following diagramcompares an uncatalyzed versus the aniline-catalyzed oxime ligationreaction (Kohler J J, ChemBioChem 2009; 10:2147-50):

However, considering the numerous health risks associated with aniline,alternative catalysts are desirable. The present invention providesaniline derivatives as alternative oxime ligation catalysts. Suchaniline derivatives include, but are not limited to, o-amino benzoicacid, m-amino benzoic acid, p-amino benzoic acid, sulfanilic acid,o-aminobenzamide, o-toluidine, m-toluidine, p-toluidine, o-anisidine,m-anisidine, and p-anisidine.

In one embodiment of the invention, m-toluidine (aka meta-toluidine,m-methylaniline, 3-methylaniline, or 3-amino-1-methylbenzene) is used tocatalyze the conjugation reactions described herein. M-toluidine andaniline have similar physical properties and essentially the same pKavalue (m-toluidine: pKa 4.73, aniline: pKa 4.63).

The nucleophilic catalysts of the invention are useful for oximeligation (e.g, using aminooxy linkage) or hydrazone formation (e.g.,using hydrazide chemistry). In various embodiments of the invention, thenucleophilic catalyst is provided in the conjugation reaction at aconcentration of 0.1, 0.2, 0.3, 0.5, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5,2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5,9.0, 9.5, 10.0, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40,45, or 50 mM. In one embodiment, the nucleophilic catalyst is providedbetween 1 to 10 mM. In various embodiments of the invention, the pHrange of conjugation reaction is 4.5, 5.0, 5.5, 6.0, 6.5, 7.0 and 7.5.In one embodiment, the pH is between 5.5 to 6.5.

Purification of Conjugated Proteins

In various embodiments, purification of a protein that has beenincubated with an oxidizing agent and/or a therapeutic protein that hasbeen conjugated with a water soluble polymer according to the presentdisclosure, is desired. Numerous purification techniques are known inthe art and include, without limitation, chromatographic methods such asion-exchange chromatography, hydrophobic interaction chromatography,size exclusion chromatography and affinity chromatography orcombinations thereof, filtration methods (e.g., UF/DF), andprecipitation methods as well as dialysis procedures and anycombinations of the aforementioned techniques (Guide to ProteinPurification, Meth. Enzymology Vol 463 (edited by Burgess R R andDeutscher M P), 2^(nd) edition, Academic Press 2009).

The following examples are not intended to be limiting but onlyexemplary of specific embodiments of the invention.

EXAMPLES Example 1 Preparation of the Homobifunctional LinkerNH₂[OCH₂CH₂]₂ONH₂

The homobifunctional linker NH₂[OCH₂CH₂]₂ONH₂

(3-oxa-pentane-1,5-dioxyamine) containing two active aminooxy groups wassynthesized according to Boturyn et al. (Tetrahedron 1997; 53:5485-92)in a two step organic reaction employing a modified Gabriel-Synthesis ofprimary amines (FIG. 3). In the first step, one molecule of2,2-chlorodiethylether was reacted with two molecules ofEndo-N-hydroxy-5-norbornene-2,3-dicarboximide in dimethylformamide(DMF). The desired homobifunctional product was prepared from theresulting intermediate by hydrazinolysis in ethanol.

Example 2 Preparation of the Homobifunctional Linker NH₂[OCH₂CH₂]₄ONH₂

The homobifunctional linker NH₂[OCH₂CH₂]₄ONH₂

(3,6,9-trioxa-undecane-1,11-dioxyamine) containing two active aminooxygroups was synthesized according to Boturyn et al. (Tetrahedron 1997;53:5485-92) in a two step organic reaction employing a modifiedGabriel-Synthesis of primary amines (FIG. 3). In the first step onemolecule of Bis-(2-(2-chlorethoxy)-ethyl)-ether was reacted with twomolecules of Endo-N-hydroxy-5-norbornene-2,3-dicarboximide in DMF. Thedesired homobifunctional product was prepared from the resultingintermediate by hydrazinolysis in ethanol.

Example 3 Preparation of the Homobifunctional Linker NH₂[OCH₂CH₂]₆ONH₂

The homobifunctional linker NH₂[OCH₂CH₂]₆ONH₂

(3,6,9,12,15-penatoxa-heptadecane-1,17-dioxyamine) containing two activeaminooxy groups was synthesized according to Boturyn et al. (Tetrahedron1997; 53:5485-92) in a two step organic reaction employing a modifiedGabriel-Synthesis of primary amines. In the first step one molecule ofhexaethylene glycol dichloride was reacted with two molecules ofEndo-N-hydroxy-5-norbornene-2,3-dicarboximide in DMF. The desiredhomobifunctional product was prepared from the resulting intermediate byhydrazinolysis in ethanol.

Example 4 Detailed Synthesis of the Aminooxy-PSA Reagent

3-oxa-pentane-1,5 dioxyamine was synthesized according to Botyryn et al(Tetrahedron 1997; 53:5485-92) in a two step organic synthesis asoutlined in Example 1.

Step 1:

To a solution of Endo-N-hydroxy-5-norbonene-2,3-dicarboxiimide (59.0 g;1.00 eq) in 700 ml anhydrous N,N-dimethylformamide anhydrous K₂CO₃(45.51 g; 1.00 eq) and 2,2-dichlorodiethylether (15.84 ml; 0.41 eq) wereadded. The reaction mixture was stirred for 22 h at 50° C. The mixturewas evaporated to dryness under reduced pressure. The residue wassuspended in 2 L dichloromethane and extracted two times with saturatedaqueous NaCl-solution (each 1 L). The Dichloromethane layer was driedover Na₂SO₄ and then evaporated to dryness under reduced pressure anddried in high vacuum to give 64.5 g of3-oxapentane-1,5-dioxy-endo-2′,3′-dicarboxydiimidenorbornene as awhite-yellow solid (intermediate 1).

Step 2:

To a solution of intermediate 1 (64.25 g; 1.00 eq) in 800 ml anhydrousEthanol, 31.0 ml Hydrazine hydrate (4.26 eq) were added. The reactionmixture was then refluxed for 2 hrs. The mixture was concentrated to thehalf of the starting volume by evaporating the solvent under reducedpressure. The occurring precipitate was filtered off. The remainingethanol layer was evaporated to dryness under reduced pressure. Theresidue containing the crude product 3-oxa-pentane-1,5-dioxyamine wasdried in vacuum to yield 46.3 g. The crude product was further purifiedby column chromatography (Silicagel 60; isocratic elution withDichloromethane/Methanol mixture, 9/1) to yield 11.7 g of the pure finalproduct 3-oxa-pentane-1,5-dioxyamine.

Example 5 Preparation of Aminooxy-PSA

1000 mg of oxidized PSA (MW=20 kD) obtained from the Serum Institute ofIndia (Pune, India) was dissolved in 16 ml 50 mM phosphate buffer pH6.0. Then 170 mg 3-oxa-pentane-1,5-dioxyamine was given to the reactionmixture. After shaking for 2 hrs at RT 78.5 mg sodium cyanoborohydridewas added and the reaction was performed for 18 hours over night. Thereaction mixture was then subjected to an ultrafiltration/diafiltrationprocedure (UF/DF) using a membrane with a 5 kD cut-off made ofregenerated cellulose (50 cm², Millipore).

Example 6 Preparation of Aminooxy-PSA Employing a ChromatographicPurification Step

1290 mg of oxidized PSA (MW=20 kD) obtained from the Serum Institute ofIndia (Pune, India) was dissolved in 25 ml 50 mM phosphate buffer pH 6.0(Buffer A). Then 209 mg 3-oxa-pentane-1,5-dioxyamine was given to thereaction mixture. After shaking for 1 h at RT 101 mg sodiumcyanoborohydride was added and the reaction was performed for 3 hours.Then the mixture was then subjected to a weak anion exchangechromatography step employing a Fractogel EMD DEAE 650-M chromatographygel (column dimension: XK26/135). The reaction mixture was diluted with110 ml Buffer A and loaded onto the DEAE column pre-equilibrated withBuffer A at a flow rate of 1 cm/min. Then the column was washed with 20CV Buffer B (20 mM Hepes, pH 6.0) to remove free3-oxa-pentane-1,5-dioxyamine and cyanide at a flow rate of 2 cm/min. Theaminooxy-PSA reagent was then eluted with a step gradient consisting of67% Buffer B and 43% Buffer C (20 mM Hepes, 1M NaCl, pH 7.5). The eluatewas concentrated by UF/DF using a 5 kD membrane made of polyethersulfone (50 cm², Millipore). The final diafiltration step was performedagainst Buffer D (20 mM Hepes, 90 mM NaCl, pH 7.4). The preparation wasanalytically characterized by measuring total PSA (Resorcinol assay) andtotal aminooxy groups (TNBS assay) to determine the degree ofmodification. Furthermore the polydispersity as well as free3-oxa-pentane-1,5-dioxyamine and cyanide was determined.

Example 7 Preparation of Aminooxy-PSA without a Reduction Step

573 mg of oxidized PSA (MW=20 kD) obtained from the Serum Institute ofIndia (Pune, India) was dissolved in 11.3 ml 50 mM phosphate buffer pH6.0 (Buffer A). Then 94 mg 3-oxa-pentane-1,5-dioxyamine was given to thereaction mixture. After shaking for 5 h at RT the mixture was thensubjected to a weak anion exchange chromatography step employing aFractogel EMD DEAE 650-M chromatography gel (column dimension:XK16/105). The reaction mixture was diluted with 50 ml Buffer A andloaded onto the DEAE column pre-equilibrated with Buffer A at a flowrate of 1 cm/min. Then the column was washed with 20 CV Buffer B (20 mMHepes, pH 6.0) to remove free 3-oxa-pentane-1,5-dioxyamine and cyanideat a flow rate of 2 cm/min. The aminooxy-PSA reagent was the eluted witha step gradient consisting of 67% Buffer B and 43% Buffer C (20 mMHepes, 1 M NaCl, pH 7.5). The eluate was concentrated by UF/DF using a 5kD membrane made of polyether sulfone (50 cm², Millipore). The finaldiafiltration step was performed against Buffer D (20 mM Hepes, 90 mMNaCl, pH 7.4). The preparation was analytically characterized bymeasuring total PSA (Resorcinol assay) and total aminooxy groups (TNBSassay) to determine the degree of modification. Furthermore thepolydispersity as well as free 3-oxa-pentane-1,5-dioxyamine wasdetermined.

Example 8 Preparation of Aminooxy-PSA without a Reduction Step in thePresence of the Nucleophilic Catalyst m-Toluidine

573 mg of oxidized PSA (MW=20 kD) obtained from the Serum Institute ofIndia (Pune, India) is dissolved in 9 ml 50 mM phosphate buffer pH 6.0(Buffer A). Then 94 mg 3-oxa-pentane-1,5-dioxyamine is given to thissolution. Subsequently 2.3 ml of a 50 mM m-toluidine stock solution areadded to this reaction mixture. After shaking for 2 h at RT the mixtureis then subjected to a weak anion exchange chromatography step employinga Fractogel EMD DEAE 650-M chromatography gel (column dimension:XK16/105). The reaction mixture is diluted with 50 ml Buffer A andloaded onto the DEAE column pre-equilibrated with Buffer A at a flowrate of 1 cm/min. Then the column is washed with 20CV Buffer B (20 mMHepes, pH 6.0) to remove free 3-oxa-pentane-1,5-dioxyamine and cyanideat a flow rate of 2 cm/min. The aminooxy-PSA reagent is the eluted witha step gradient consisting of 67% Buffer B and 43% Buffer C (20 mMHepes, 1 M NaCl, pH 7.5). The eluate is concentrated by UF/DF using a 5kD membrane made of polyether sulfone (50 cm², Millipore). The finaldiafiltration step is performed against Buffer D (20 mM Hepes, 90 mMNaCl, pH 7.4). The preparation is analytically characterized bymeasuring total PSA (Resorcinol assay) and total aminooxy groups (TNBSassay) to determine the degree of modification. Furthermore thepolydispersity as well as free 3-oxa-pentane-1,5-dioxyamine isdetermined.

Example 9 Preparation of Aminooxy-PSA Reagent

An Aminooxy-PSA reagent was prepared according to the Examples 4-8.After diafiltration, the product was frozen at −80° C. and lyophilized.After lyophilization the reagent was dissolved in the appropriate volumeof water and used for preparation of PSA-protein conjugates viacarbohydrate modification.

Example 10 Evaluation of the Efficacy of Different AlternativeNucleophilic Catalysts

rFIX was incubated with sodium periodate, aminooxy-PSA reagent understandardized conditions (1 mg/ml rFIX in 20 mM L-histidine, 150 mM NaCl,5 mM CaCl₂, pH 6.0, 5-fold molar aminooxy-PSA reagent excess, 100 μMNaIO₄) using different nucleophilic catalysts (aniline, m-toluidine,o-anisidine, m-anisidine, o-aminobenzoic acid, m-aminobenzoic acid,p-aminobenzoic acid, p-aminobenzamide, sulfanilic acid/standardconcentration: 10 mM) The reaction was carried out for 2 hrs in the darkat room temperature under gentle stirring and quenched for 15 min atroom temperature by the addition of aqueous cysteine solution with afinal concentration of 1 mM.

The coupling efficiency was determined by SDS-PAGE using an InvitrogenX-cell mini system. Samples were spiked with lithium dodecyl sulfate(LDS) buffer and denatured for 10 min at 70° C. Then the samples wereapplied on 3-8% TRIS-acetate gels and ran at 150 V for 60 min.Subsequently the gels were stained with Coomassie.

In addition the samples were characterized by use of a SEC-HPLC systemusing a Agilent 1200 HPLC system equipped with a Shodex KW 803 columnunder conditions as previously described (Kolarich et al, Transfusion2006; 46:1959-77).

50 μl of samples were injected undiluted and eluted isocratically with a0.22 μm filtered solution of 20 mM NaH₂PO₄, 50 mM Na₂SO₄, pH 6.1 at aflow rate of 0.5 ml/min. The elution pattern was recorded at 280 nm.

The results are summarized in FIGS. 5A-C and 6 (SDS PAGE) and Table 2(SEC-HPLC results). The catalytic effect of the different preparationsis demonstrated. It is shown that the use of m-toluidine leads toequivalent results as obtained with aniline.

TABLE 2 di-PSAylated mono- free nucleophilic catalysts rFIX PSAylatedrFIX rFIX no catalyst 4.5% 24.9% 70.6% 10 mM aniline 47.7% 33.6% 18.7%10 mM m-toluidine 31.4% 40.8% 27.8% 10 mM o-aminobenzioc acid 30.9%38.5% 30.6% 10 mM m-aminobenzioc acid 27.6% 38.0% 34.4% 10 mMp-aminobenzioc acid 18.1% 39.3% 42.6% 10 mM o-aminobenzamide 15.9% 38.4%45.7% 10 mM sulfanilic acid 11.8% 35.8% 52.4%

Example 11 Polysialylation of rFIX Using Aminooxy-PSA and m-Toluidine asa Nucleophilic Catalyst

Method 1:

12.3 mg rFIX was dissolved in 6.1 ml histidine buffer, pH 6.0 (20 mML-histidine, 150 mM NaCl, 5 mM CaCl₂). 254 μl of an aqueous sodiumperiodate solution (5 mM) was then added and the reaction mixture isincubated for 1 h in the dark at 4° C. under gentle stirring andquenched for 15 min at room temperature by the addition of 6.5 μl of a 1M aqueous cysteine solution. The mixture was subsequently subjected toUF/DF employing Vivaspin 15R 10 kD centrifugal filtrators to removeexcess periodate, quencher and the byproducts thereof.

The retentate (8.8 ml), containing oxidized rFIX was mixed with 2.46 mlof an aqueous m-toluidine solution (50 mM) and incubated for 30 min atroom temperature. Then aminooxy-PSA reagent with a MW of 20 kD(described above) was added to give a 5-fold molar reagent excess. Thismixture was incubated for 2.5 h at RT in the dark under gentle stirring.

The free rFIX was removed by means of anion exchange chromatography(AEC). The reaction mixture was diluted with 15 ml Buffer A (50 mMHepes, 5 mM CaCl₂, pH 7.5) and loaded onto a 20 ml HiPrep QFF 16/10column (GE Healthcare, Fairfield, Conn.) pre-equilibrated with Buffer A.The column was then eluted with Buffer B (50 mM Hepes, 1 M NaCl, 5 mMCaCl₂, pH 7.5). Free rFIX elutes at a conductivity between 12-25 mS/cmand the conjugate between 27-45 mS/cm. The conductivity of the conjugatecontaining fractions was subsequently raised to 190 mS/cm with Buffer C(50 mM Hepes, 5M NaCl, 5 mM CaCl₂, pH 6.9) and loaded onto a 20 mlHiPrep Butyl FF 16/10 column (GE Healthcare, Fairfield, Conn.)pre-equilibrated with Buffer D (50 mM Hepes, 3 M NaCl, 5 mM CaCl₂, pH6.9). Free aminooxy-PSA reagent was washed out within 5 CV Buffer D.Subsequently the conjugate is eluted with 100% Buffer E (50 mM Hepes, 5mM CaCl₂, pH 7.4). The conjugate containing fractions were concentratedby UF/DF using Vivaspin 15R 10 kD centrifugal filtrator. The finaldiafiltration step was performed against histidine buffer, Ph 7.2containing 150 mM NaCl and 5 mM CaCl₂. The preparation was analyticallycharacterized by measuring total protein (Bradford) and FIX chromogenicactivity. The PSA-rFIX conjugate showed a specific activity of >50% incomparison to native rFIX is determined.

Method 2:

12.3 mg rFIX is dissolved in L-histidine buffer, pH 6.0 (20 mML-histidine, 150 mM NaCl, 5 mM CaCl₂) to get a final proteinconcentration of 1 mg rFIX/ml. A 5 mM aqueous sodium periodate solutionis added to get a final concentration of 100 μM and the reaction mixtureis incubated for 1 hour in the dark at 4° C. under gentle stirring at pH6.0 and quenched for 15 min at room temperature by the addition of an 1M aqueous L-cysteine solution (or other quenching reagents) to get afinal concentration of 10 mM. The mixture is subsequently subjected toUF/DF employing Vivaspin 15R 10 kD centrifugal filtrators to removeexcess periodate, quencher and the byproducts thereof.

The obtained retentate (8.8 ml), containing oxidized rFIX, is mixed withan aqueous m-toluidine solution (50 mM) to give a final concentration of10 mM and incubated for 30 min at room temperature. Then aminooxy-PSAreagent with a MW of 20 kD (described above) is added to give a 5-foldmolar reagent excess. This mixture was incubated at pH 6.0 for 2.5 hoursat room temperature; 0.5 hours to 18 hours at +4° C.) in the dark undergentle stirring.

The free rFIX is removed by means of anion exchange chromatography(AEC). The reaction mixture is diluted with appropriate amounts ofBuffer A (50 mM Hepes, 5 mM CaCl₂, pH 7.5) to correct the solutionsconductivity and pH prior to load onto a 20 ml HiPrep QFF 16/10 column(GE Healthcare, Fairfield, Conn.) pre-equilibrated with buffer A. Thenthe column is eluted with Buffer B (50 mM Hepes, 1 M NaCl, 5 mM CaCl₂,pH 7.5). Free rFIX is eluted by a step gradient using 25% of Buffer B,which results in a conductivity between 12-25 mS/cm in the obtainedfraction and the conjugate using a step gradient of 50% Buffer B, whichresults in a conductivity between 27-45 mS/cm in the conjugate fraction.The conductivity of the conjugate containing fraction is subsequentlyraised to 190 mS/cm with Buffer C (50 mM Hepes, 5 M NaCl, 5 mM CaCl₂, pH6.9 or by use of anti-chaotropic salts e.g. ammonium sulphate, ammoniumacetate etc.) and loaded onto a 20 ml HiPrep Butyl FF 16/10 column (GEHealthcare, Fairfield, Conn. or comparable HIC media) pre-equilibratedwith Buffer D (50 mM Hepes, 3 M NaCl, 5 mM CaCl₂, pH 6.9). Freeaminooxy-PSA reagent is washed out within 5 CV Buffer D. Subsequently,the conjugate is eluted with 100% Buffer E (50 mM Hepes, 5 mM CaCl₂, pH7.4). The conjugate containing fractions are concentrated by UF/DF usinga 10 kD membrane made of regenerated cellulose (88 cm2, cut-off 10 kD,Millipore). The final diafiltration step is performed againstL-histidine buffer, pH 7.2 containing 150 mM NaCl and 5 mM CaCl₂. Thepreparation is analytically characterized by measuring total protein(Bradford and BCA procedure) and FIX chromogenic- and clotting activity.For the PSA-rFIX conjugate a specific activity of >50% in comparison tonative rFIX is determined.

Method 3:

25.4 mg rFIX was dissolved in 18.7 ml histidine buffer, pH 6.0 (20 mML-histidine, 150 mM NaCl, 5 mM CaCl₂). 531 μl of an aqueous sodiumperiodate solution (5 mM) and 5.07 ml of an aqueous m-toluidine solution(50 mM) were then added. Subsequently, the aminooxy-PSA reagent with aMW of 20 kD (described above) was added to give a 5-fold molar reagentexcess. The mixture was incubated for 2 h in the dark at roomtemperature under gentle stirring and quenched for 15 min at roomtemperature by the addition of 25 μl of 1 M aqueous cysteine solution.

The free rFIX was removed by means of anion exchange chromatography(AEC). The reaction mixture was diluted with 20 ml Buffer A (50 mMHepes, 5 mM CaCl₂, pH 7.5) and loaded onto a 20 ml HiPrep QFF 16/10column (GE Healthcare, Fairfield, Conn.) pre-equilibrated with Buffer A.Then the column was eluted with Buffer B (50 mM Hepes, 1 M NaCl, 5 mMCaCl₂, pH 7.5). Free rFIX eluted at a conductivity between 12-25 mS/cmand the conjugate between 27-45 mS/cm. The conductivity of the conjugatecontaining fractions was subsequently raised to 190 mS/cm with Buffer C(50 mM Hepes, 5 M NaCl, 5 mM CaCl₂, pH 6.9) and loaded onto a 20 mlHiPrep Butyl FF 16/10 column (GE Healthcare, Fairfield, Conn.)pre-equilibrated with Buffer D (50 mM Hepes, 3 M NaCl, 5 mM CaCl₂, pH6.9). Free aminooxy-PSA reagent was washed out within 5 CV Buffer D.Subsequently, the conjugate was eluted with 100% Buffer E (50 mM Hepes,5 mM CaCl₂, pH 7.4). The conjugate containing fractions wereconcentrated by UF/DF using a 10 kD membrane made of regeneratedcellulose (88 cm2, cut-off 10 kD, Millipore). The final diafiltrationstep was performed against histidine buffer, pH 7.2 containing 150 mMNaCl and 5 mM CaCl₂. The preparation was analytically characterized bymeasuring total protein (Bradford) and FIX chromogenic activity. For thePSA-rFIX conjugate a specific activity of >50% in comparison to nativerFIX was determined. The conjugate was additionally analyticallycharacterized by Size Exclusion HPLC using a Agilent 1200 HPLC systemequipped with a Shodex KW 803 column under conditions as previouslydescribed (Kolarich et al, Transfusion 2006; 46:1959-77). It was shownthat the preparation contains no free FIX. The conjugate consisted of57% mono-polysialylated and 31% di-polysialylated and 12%tri-polysialyated product.

Method 4:

25.4 mg rFIX was dissolved in L-histidine buffer, pH 6.0 (20 mML-histidine, 150 mM NaCl, 5 mM CaCl₂) to get a final proteinconcentration of 2 mg rFIX/ml. Subsequently an 5 mM aqueous sodiumperiodate solution was added within 15 minutes to give a finalconcentration of 100 μM, followed by addition of an 50 mM aqueousm-toluidine solution to get a final concentration of 10 mM within a timeperiod of 30 minutes. Then the aminooxy-PSA reagent with a MW of 20 kD(described above) was added to give a 5-fold molar reagent excess. Aftercorrection of the pH to 6.0 the mixture was incubated for 2 h in thedark at room temperature under gentle stirring and quenched for 15 minat room temperature by the addition of a 1 M aqueous L-cysteine solutionto give a final concentration of 10 mM.

The free rFIX was removed by means of ion exchange chromatography (IEC).The reaction mixture was diluted with appropriate amounts of Buffer A(50 mM Hepes, 5 mM CaCl₂, pH 7.5) to correct the solutions conductivityand pH value prior to load onto a 20 ml HiPrep QFF 16/10 column (GEHealthcare, Fairfield, Conn.) pre-equilibrated with Buffer A. Then thecolumn was eluted with Buffer B (50 mM Hepes, 1 M NaCl, 5 mM CaCl₂, pH7.5). Free rFIX was eluted by a step gradient using 25% of Buffer B,which results in a conductivity between 12-25 mS/cm in the obtainedfraction and the conjugate using a step gradient of 50% Buffer B, whichresults in a conductivity between 27-45 mS/cm in the conjugate fraction.The conductivity of the conjugate containing fraction was subsequentlyraised to 190 mS/cm with Buffer C (50 mM Hepes, 5 M NaCl, 5 mM CaCl₂, pH6.9; by use of anti-chaotropic salts e.g. ammonium acetate) and loadedonto a 20 ml HiPrep Butyl FF 16/10 column (GE Healthcare, Fairfield,Conn.; or comparable HIC media) pre-equilibrated with Buffer D (50 mMHepes, 3 M NaCl, 5 mM CaCl₂, pH 6.9). Free aminooxy-PSA reagent waswashed out within 5 CV Buffer D. Subsequently the conjugate was elutedwith 100% Buffer E (50 mM Hepes, 5 mM CaCl₂, pH 7.4). The conjugatecontaining fractions were concentrated by UF/DF using a 10 kD membranemade of regenerated cellulose (88 cm2, cut-off 10 kD, Millipore). Thefinal diafiltration step was performed against L-histidine buffer, pH7.2 containing 150 mM NaCl and 5 mM CaCl₂. The preparation wasanalytically characterized by measuring total protein (Bradford and BCAprocedure) and FIX chromogenic- and clotting activity. For the PSA-rFIXconjugate a specific activity of >50% in comparison to native rFIX wasdetermined. The conjugate was additionally analytically characterized bySize Exclusion HPLC using a Agilent 1200 HPLC system equipped with aShodex KW 803 column under conditions as previously described (Kolarichet al, Transfusion 2006; 46:1959-77). It was shown that the preparationcontains no free FIX. The conjugate consisted of 57% mono-polysialylatedand 31% di-polysialylated and 12% tri-polysialyated product.

Example 12 Polysialylation of rFVIII Using Aminooxy-PSA and m-Toluidineas a Nucleophilic Catalyst

Method 1:

50 mg rFVIII was transferred into reaction buffer (50 mM Hepes, 350 mMsodium chloride, 5 mM calcium chloride, pH 6.0) and diluted to obtain aprotein concentration of 1 mg/ml. To this solution, NaIO₄ was added togive a final concentration of 200 μM. The oxidation was carried at RTfor 30 min in the dark under gentle shaking. Then the reaction wasquenched with cysteine (final concentration: 10 mM) for 60 min at RT.The solution was subjected to an IEX column with a volume of 20 ml(Merck EMD TMAE (M)) which was equilibrated with Buffer A (20 mM Hepes,5 mM CaCl₂, pH 7.0). The column was equilibrated with 5 CV Buffer A.Then the oxidized rFVIII was eluted with Buffer B (20 mM Hepes, 5 mMCaCl₂, 1M NaCl, pH 7.0). The rFVIII containing fractions were collected.The protein content was determined (Coomassie, Bradford) and adjusted to1 mg/ml with reaction buffer and adjusted to pH 6.0 by dropwise additionof 0.5 M HCl. Then a 50-fold molar excess of a aminooxy-PSA reagent witha MW of 20 kD (described above) was added followed by m-toluidine as anucleophilic catalyst (final concentration: 10 mM). The couplingreaction was performed for 2 hours in the dark under gentle shaking atroom temperature. The excess of aminooxy-PSA reagent was removed bymeans of HIC. The conductivity of the reaction mixture was raised to 130mS/cm by adding a buffer containing ammonium acetate (50 mM Hepes, 350mM sodium chloride, 5 mM calcium chloride, 8 M ammonium acetate, pH 6.9)and loaded onto a column filled with 80 ml Phenyl Sepharose FF (GEHealthcare, Fairfield, Conn.) pre-equilibrated with 50 mM Hepes, 2.5 Mammonium acetate, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.9.Subsequently, the conjugate was eluted with 50 mM Hepes buffer pH 7.5containing 5 mM CaCl₂. Finally, the PSA-rFVIII containing fractions werecollected and subjected to UF/DF by use of a 30 kD membrane made ofregenerated cellulose (88 cm², Millipore). The preparation wasanalytically characterized by measuring total protein (Coomassie,Bradford) and FVIII chromogenic activity. The PSA-rFVIII conjugateshowed a specific activity of >70% in comparison to native rFVIII wasdetermined.

Method 2:

58 mg of recombinant factor VIII (rFVIII) derived from the ADVATEprocess in Hepes buffer (50 mM HEPES, ˜350 mM sodium chloride, 5 mMcalcium chloride, 0.1% Polysorbate 80, pH 7.4) is dissolved in reactionbuffer (50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH6.0) to get a final protein concentration of 1.0+/−0.25 mg/ml. Then thepH of the solution is corrected to 6.0 by drop wise addition of a 0.5 Naqueous HCl solution. Subsequently, a 40 mM aqueous sodium periodatesolution is added within 10 minutes to give a concentration of 200 μM.The oxidation reaction is carried out for 30+/−5 min at a temperature(T) of T=+22+/−2° C. Then the reaction is stopped by addition of anaqueous L-cysteine solution (1 M) within 15 minutes at T=+22+/−2° C. togive a final concentration of 10 mM in the reaction mixture andincubation for 60+/−5 min.

The oxidized rFVIII is further purified by anion exchange chromatographyon EMD TMAE (M) (Merck). The mixture is diluted with Buffer A (20 mMHepes, 5 mM CaCl₂, pH 6.5) to give a conductivity of 5 ms/cm. Thissolution is loaded onto the IEX column (bed height: 5.4 cm) with acolumn volume of 10 ml using a flow rate of 1.5 cm/min. This column issubsequently washed (flow rate: 1.5 cm/min) with 5 CV of a 92:8 mixture(w/w) of Buffer A and Buffer B (20 mM Hepes, 5 mM CaCl₂, 1.0 M NaCl, pH7.0). Then the oxidized rFVIII is eluted with a 50:50 (w/w) mixture ofBuffer A and Buffer B followed by a postelution step with 5 CV of BufferB. The elution steps are carried out by use of a flow rate of 1.0cm/min.

Subsequently, the aminooxy-polysialic acid (PSA-ONH₂) reagent is addedin a 50-fold molar excess to the eluate containing the purified oxidizedrFVIII within a maximum time period (t) of 15 minutes under gentlestirring. Then an aqueous m-toluidine solution (50 mM) is added within15 minutes to get a final concentration of 10 mM. The reaction mixtureis incubated for 120+/−10 min. in the dark at a temperature (T) ofT=+22+/−2° C. under gentle shaking.

The obtained PSA-rFVIII conjugate is purified by Hydrophobic InteractionChromatography (HIC) using a Phenyl Sepharose FF low sub resin (GEHealthcare) packed into a column manufactured by GE Healthcare with abed height (h) of 15 cm and a resulting column volume (CV) of 81 ml.

The reaction mixture is spiked with ammonium acetate by addition of 50mM Hepes buffer, containing 350 mM sodium chloride, 8 M ammoniumacetate, 5 mM calcium chloride, pH 6.9. Two volumes of the reactionmixture are mixed with 1 volume of the ammonium acetate containingbuffer system and the pH value is corrected to pH 6.9 by drop wiseaddition of a 0.5 N aqueous NaOH solution. This mixture is loaded ontothe HIC column at flow rate of 1 cm/min followed by a washing stepusing >3 CV equilibration buffer (50 mM Hepes, 350 mM sodium chloride,2.5 M ammonium acetate, 5 mM calcium chloride, pH 6.9).

For removal of reaction by-products and anti-chaotropic salt a secondwashing step is performed with >5 CV washing buffer 1 (50 mM Hepes, 3 Msodium chloride, 5 mM calcium chloride, pH 6.9) in upflow mode at a flowrate of 2 cm/min. Then elution of purified PSA-rFVIII conjugate isperformed in down flow mode using a step gradient of 40% washing buffer2 (50 mM Hepes, 1.5 M sodium chloride, 5 mM calcium chloride, pH 6.9)and 60% elution buffer (20 mM Hepes, 5 mM calcium chloride, pH 7.5) at aflow rate of 1 cm/min. The elution of the PSA-rFVIII conjugate ismonitored at UV 280 nm and the eluate containing the conjugate iscollected within <4 CV. The post elution step is performed with >3 CVelution buffer under the same conditions to separate minor and/or nonmodified rFVIII from the main product.

Finally the purified conjugate is concentrated by ultra-/diafiltration(UF/DF) using a membrane made of regenerated cellulose with a molecularweight cut off 30 kD (88 cm², Millipore).

The conjugate prepared by use of this procedure are analyticallycharacterized by measuring total protein, FVIII chromogenic activity anddetermination of the polysialyation degree by measuring the PSA content(resorcinol assay). For the conjugate obtained a specific activity >50%and a PSA degree >5.0 is calculated.

Method 3:

50 mg rFVIII was transferred into reaction buffer (50 mM Hepes, 350 mMsodium chloride, 5 mM calcium chloride, pH 6.0) and diluted to obtain aprotein concentration of 1 mg/ml. A 50-fold molar excess of aminooxy-PSAreagent with a MW of 20 kD (described above) was added followed bym-toluidine as a nucleophilic catalyst (final concentration: 10 mM) andNaIO₄ (final concentration: 400 μM). The coupling reaction was performedfor 2 hours in the dark under gentle shaking at room temperature.Subsequently, the reaction was quenched with cysteine for 60 min at RT(final concentration: 10 mM). Then the conductivity of the reactionmixture was raised to 130 mS/cm by adding a buffer containing ammoniumacetate (50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, 8 Mammonium acetate, pH 6.9) and loaded onto a column filled with 80 mlPhenyl Sepharose FF (GE Healthcare, Fairfield, Conn.) pre-equilibratedwith 50 mM Hepes, 2.5 M ammonium acetate, 350 mM sodium chloride, 5 mMcalcium chloride, 0.01% Tween 80, pH 6.9. Subsequently, the conjugatewas eluted with 50 mM Hepes, 5 mM calcium chloride, pH 7.5. Finally, thePSA-rFVIII containing fractions were collected and subjected to UF/DF byuse of a 30 kD membrane made of regenerated cellulose (88 cm²,Millipore). The preparation was analytically characterized by measuringtotal protein (Bradford) and FVIII chromogenic activity. For thePSA-rFVIII conjugate a specific activity of ≧70% in comparison to nativerFVIII was determined.

Method 4:

50 mg recombinant factor VIII (rFVIII) derived from the ADVATE processin 50 mM Hepes buffer (50 mM HEPES, ˜350 mM sodium chloride, 5 mMcalcium chloride, 0.1% Polysorbate 80, pH 7.4) was dissolved in reactionbuffer (50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH6.0) to get a final protein concentration of 1.0+/−0.25 mg/ml. Then thepH of the solution was corrected to 6.0 by drop wise addition of a 0.5 Naqueous HCl solution.

Subsequently, the aminooxy-polysialic acid (PSA-ONH₂) reagent was addedin a 50-fold molar excess to this rFVIII solution within a maximum timeperiod (t) of 15 minutes under gentle stirring. Then an aqueousm-toluidine solution (50 mM) was added within 15 minutes to get a finalconcentration of 10 mM. Finally, a 40 mM aqueous sodium periodatesolution was added to give a concentration of 400 μM.

The reaction mixture was incubated for 120+/−10 min. in the dark at atemperature (T) of T=+22+/−2° C. under gentle shaking. Then the reactionwas stopped by the addition of an aqueous L-cysteine solution (1 M) togive a final concentration of 10 mM in the reaction mixture andincubation for 60+/−5 min.

The obtained PSA-rFVIII conjugate was purified by HydrophobicInteraction Chromatography (HIC) using a Phenyl Sepharose FF low subresin (GE Healthcare) packed into a column manufactured by GE Healthcarewith a bed height (h) of 15 cm and a resulting column volume (CV) of 81ml.

The reaction mixture was spiked with ammonium acetate by addition of 50mM Hepes buffer, containing 350 mM sodium chloride, 8 M ammoniumacetate, 5 mM calcium chloride, pH 6.9. Two volumes of the reactionmixture was mixed with 1 volume of the ammonium acetate containingbuffer system and the pH value was corrected to pH 6.9 by drop wiseaddition of an 0.5 N aqueous NaOH solution. This mixture was loaded ontothe HIC column using a flow rate of 1 cm/min followed by a washing stepusing >3 CV equilibration buffer (50 mM Hepes, 350 mM sodium chloride,2.5 M ammonium acetate, 5 mM calcium chloride, pH 6.9).

For removal of reaction by-products and anti-chaotropic salt a secondwashing step was performed with >5CV washing buffer 1 (50 mM Hepes, 3 Msodium chloride, 5 mM calcium chloride, pH 6.9) in upflow mode at a flowrate of 2 cm/min. Then elution of purified rFVIII conjugate wasperformed in down flow mode using a step gradient of 40% washing buffer2 (50 mM Hepes, 1.5 M sodium chloride, 5 mM calcium chloride, pH 6.9)and 60% elution buffer (20 mM Hepes, 5 mM calcium chloride, pH 7.5) at aflow rate of 1 cm/min. The elution of the PSA-rFVIII conjugate wasmonitored at UV 280 nm and the eluate containing the conjugate wascollected within <4 CV. The post elution step was performed with >3 CVelution buffer under the same conditions to separate minor and/or nonmodified rFVIII from the main product.

Finally, the purified conjugate was concentrated by ultra-/diafiltration(UF/DF) using a membrane made of regenerated cellulose with a molecularweight cut off 30 kD (88 cm², Millipore).

The conjugates prepared by use of this procedure were analyticallycharacterized by measuring total protein, FVIII chromogenic activity anddetermination of the polysialyation degree by measuring the PSA content(resorcinol assay).

-   -   Analytical data (mean of 6 consecutive batches):    -   Process yield (Bradford): 58.9%    -   Process yield (FVIII chrom.): 46.4%    -   Specific activity: (FVIII chrom./mg protein): 4148 IU/mg    -   Specific activity (% of starting material): 79.9%    -   PSA degree (mol/mol): 8.1

Example 13 PEGylation of r FVIII Using an Aminooxy-PEG Reagent andm-Toluidine as a Nucleophilic Catalyst

Method 1:

rFVIII is PEGylated by use of a linear 20 kD PEGylation reagentcontaining an aminooxy group. An example of this type of reagent is theSunbright® CA series from NOF (NOF Corp., Tokyo, Japan). 14.7 mg rFVIIIis dissolved in 7.0 ml histidine buffer, pH 6.0 (20 mM L-histidine, 150mM NaCl, 5 mM CaCl₂). Then 296 μl of an aqueous sodium periodatesolution (5 mM) is added and the reaction mixture is incubated for 1 hin the dark at 4° C. under gentle stirring and quenched for 15 min atroom temperature by the addition of 7.5 μl of a 1 M aqueous cysteinesolution. The mixture was subsequently subjected to UF/DF employingVivaspin 15R 10 kD centrifugal filtrators to remove excess periodate,quencher and the byproducts thereof.

The retentate (10.9 ml), containing oxidized rFVIII, is mixed with 2.94ml of an aqueous m-toluidine solution (50 mM) and incubated for 30 minat room temperature. Then aminooxy-PEG reagent with a MW of 20 kD isadded to give a 5-fold molar reagent excess. This mixture was incubatedfor 2.5 h at room temperature in the dark under gentle stirring.

Finally, the PEG-rFVIII conjugate is purified by ion-exchangechromatography on Q Sepharose FF. 1.5 mg protein/ml gel is loaded on thecolumn equilibrated with 50 mM Hepes buffer, pH 7.4 containing 5 mMCaCl₂. The conjugate is eluted with 50 mM Hepes buffer containing 5 mMCaCl₂ and 500 mM sodium chloride, pH 7.4 and is then subjected to UF/DFusing a 30 kD membrane (50 cm2, Millipore). The preparation isanalytically characterized by measuring total protein (Coomassie,Bradford) and FVIII chromogenic activity. It is expected that thePEG-rFVIII conjugate will demonstrate a specific activity of >70% incomparison to native rFVIII was determined.

Method 2:

rFVIII is PEGylated by use of a linear 20 kD PEGylation reagentcontaining an aminooxy group. An example of this type of reagent is theSunbright® CA series from NOF (NOF Corp., Tokyo, Japan). A startingweight or concentration of rFVIII is dissolved in or transferred to areaction buffer (50 mM Hepes, 350 mM sodium chloride, 5 mM calciumchloride, pH 6.0) to get a final protein concentration of 1.0+/−0.25mg/ml. Then the pH of the solution is corrected to 6.0 by drop wiseaddition of a 0.5 N aqueous HCl solution. Subsequently a 40 mM aqueoussodium periodate solution is added within 10 minutes to give aconcentration of 200 μM. The oxidation reaction is carried out for30+/−5 min at a temperature (T) of T=+22+/−2° C. Then the reaction isstopped by addition of an aqueous L-cysteine solution (1 M) within 15minutes at T=+22+/−2° C. to give a final concentration of 10 mM in thereaction mixture and incubation for 60+/−5 min.

The oxidized rFVIII is further purified by anion exchange chromatographyon EMD TMAE (M) (Merck). The mixture is diluted with Buffer A (20 mMHepes, 5 mM CaCl₂, pH 6.5) to give a conductivity of 5 ms/cm. Thissolution is loaded onto the IEX column (bed height: 5.4 cm) with acolumn volume of 10 ml using a flow rate of 1.5 cm/min. This column issubsequently washed (flow rate: 1.5 cm/min) with 5 CV of a 92:8 mixture(w/w) of Buffer A and Buffer B (20 mM Hepes, 5 mM CaCl₂, 1.0 M NaCl, pH7.0). Then the oxidized rFVIII is eluted with a 50:50 (w/w) mixture ofBuffer A and Buffer B followed by a postelution step with 5 CV of BufferB. The elution steps are carried out by use of a flow rate of 1.0cm/min.

Subsequently, the aminooxy-PEG reagent with a MW of 20 kD reagent isadded in a 50-fold molar excess to the eluate containing the purifiedoxidized rFVIII within a maximum time period (t) of 15 minutes undergentle stirring. Then an aqueous m-toluidine solution (50 mM) is addedwithin 15 minutes to get a final concentration of 10 mM. The reactionmixture is incubated for 120+/−10 min. in the dark at a temperature (T)of T=+22+/−2° C. under gentle shaking.

The obtained PEG-rFVIII conjugate is purified by Hydrophobic InteractionChromatography (HIC) using a Phenyl Sepharose FF low sub resin (GEHealthcare) packed into a column manufactured by GE Healthcare with abed height (h) of 15 cm and a resulting column volume (CV) of 81 ml.

The reaction mixture is spiked with ammonium acetate by addition of 50mM Hepes buffer, containing 350 mM sodium chloride, 8 M ammoniumacetate, 5 mM calcium chloride, pH 6.9. Two volumes of the reactionmixture are mixed with 1 volume of the ammonium acetate containingbuffer system and the pH value is corrected to pH 6.9 by drop wiseaddition of a 0.5 N aqueous NaOH solution. This mixture is loaded ontothe HIC column using a flow rate of 1 cm/min followed by a washing stepusing >3 CV equilibration buffer (50 mM Hepes, 350 mM sodium chloride,2.5 M ammonium acetate, 5 mM calcium chloride, pH 6.9).

For removal of reaction by-products and anti-chaotropic salt a secondwashing step is performed with >5 CV washing buffer 1 (50 mM Hepes, 3 Msodium chloride, 5 mM calcium chloride, pH 6.9) in upflow mode at a flowrate of 2 cm/min. Then elution of purified rFVIII conjugate is performedin down flow mode using a step gradient of 40% washing buffer 2 (50 mMHepes, 1.5 M sodium chloride, 5 mM calcium chloride, pH 6.9) and 60%elution buffer (20 mM Hepes, 5 mM calcium chloride, pH 7.5) at a flowrate of 1 cm/min. The elution of the PEG-rFVIII conjugate is monitoredat UV 280 nm and the eluate containing the conjugate is collected within<4 CV. The post elution step is performed with >3 CV elution bufferunder the same conditions to separate minor and/or non modified rFVIIIfrom the main product.

Finally, the purified conjugate is concentrated by ultra-/diafiltration(UF/DF) using a membrane made of regenerated cellulose with a molecularweight cut off 30 kD (Millipore).

The conjugate prepared by use of this procedure are analyticallycharacterized by measuring total protein and biological activityaccording to methods known in the art.

Method 3:

rFVIII is PEGylated by use of a linear 20 kD PEGylation reagentcontaining an aminooxy group. An example of this type of reagent is theSunbright® CA series from NOF (NOF Corp., Tokyo, Japan). 7.84 mg rFVIII,dissolved in 6 ml Hepes buffer (50 mM Hepes, 150 mM sodium chloride, 5mM calcium chloride, pH 6.0) are mixed with 314 μl of an aqueous sodiumperiodate solution (10 mM), and 1.57 ml of an aqueous m-toluidinesolution (50 mM). Subsequently the aminooxy reagent is added to give a20-fold molar reagent excess. The mixture is incubated for 2 h in thedark at room temperature under gentle stirring and quenched for 15 minat room temperature by the addition of 8 μl of aqueous cysteine solution(1 M).

Finally the PEG-rFVIII conjugate is purified by ion-exchangechromatography on Q-Sepharose FF. 1.5 mg protein/ml gel is loaded on thecolumn pre equilibrated with 50 mM Hepes buffer, pH 7.4 containing 5 mMCaCl₂. The conjugate is eluted with 50 mM Hepes buffer containing 5 mMCaCl₂ and 500 mM sodium chloride, pH 7.4 and is then subjected to UF/DFusing a 30 kD membrane (88 cm², Millipore). The analyticalcharacterization of the conjugate by FVIII chromogenic assay anddetermination of total protein (Bradford) shows a specific activityof >60% compared to the rFVIII starting material.

Method 4:

rFVIII is PEGylated by use of a linear 20 kD PEGylation reagentcontaining an aminooxy group. An example of this type of reagent is theSunbright® CA series from NOF (NOF Corp., Tokyo, Japan). An initialconcentration or weight of rFVIII is transferred or dissolved in Hepesbuffer (50 mM Hepes, 150 mM sodium chloride, 5 mM calcium chloride, pH6.0) to get a final protein concentration of 2 mg rFVIII/ml.Subsequently, an 5 mM aqueous sodium periodate solution is added within15 minutes to give a final concentration of 100 μM, followed by additionof an 50 mM aqueous m-toluidine solution to get a final concentration of10 mM within a time period of 30 minutes. Then the aminooxy-PEG reagentwith a MW of 20 kD (described above) is added to give a 20-fold molarexcess. After correction of the pH to 6.0 the mixture is incubated for 2h in the dark at room temperature under gentle stirring and quenched for15 min at room temperature by the addition of a 1 M aqueous L-cysteinesolution to give a final concentration of 10 mM.

The free rFVIII is removed by means of ion exchange chromatography(IEC). The reaction mixture was diluted with appropriate amounts ofBuffer A (50 mM Hepes, 5 mM CaCl₂, pH 7.5) to correct the solutionsconductivity and pH value prior to load onto a 20 ml HiPrep QFF 16/10column (GE Healthcare, Fairfield, Conn.) pre-equilibrated with Buffer A.Then the column was eluted with Buffer B (50 mM Hepes, 1 M NaCl, 5 mMCaCl₂, pH 7.5). Free rFVIII was eluted by a step gradient using 25% ofBuffer B, which results in a conductivity between 12-25 mS/cm in theobtained fraction and the conjugate using a step gradient of 50% BufferB, which results in a conductivity between 27-45 mS/cm in the conjugatefraction. The conductivity of the conjugate containing fraction issubsequently raised with Buffer C (50 mM Hepes, 5 M NaCl, 5 mM CaCl₂, pH6.9; by use of anti-chaotropic salts e.g. ammonium acetate, ammoniumsulphate etc.) and loaded onto a 20 ml HiPrep Butyl FF 16/10 column (GEHealthcare, Fairfield, Conn.; or comparable HIC media) pre-equilibratedwith Buffer D (50 mM Hepes, 3 M NaCl, 5 mM CaCl₂, pH 6.9). FreePEG-reagent was washed out within 5 CV Buffer D. Subsequently, theconjugate was eluted with 100% Buffer E (50 mM Hepes, 5 mM CaCl₂, pH7.4). The conjugate containing fractions are concentrated by UF/DF usinga 10 kD membrane made of regenerated cellulose (88 cm2, cut-off 10 kD,Millipore). The final diafiltration step is performed against Hepesbuffer (50 mM Hepes, 5 mM CaCl₂, pH 7.5).

The preparation is analytically characterized by measuring total protein(Bradford and BCA procedure) and biological activity according to knownmethods.

Example 14 Polysialylation of rFVIIa Using Aminooxy-PSA and m-Toluidineas a Nucleophilic Catalyst

Method 1:

A starting concentration or weight of recombinant factor VIIa (rFVIIa)is transferred or dissolved in reaction buffer (50 mM Hepes, 350 mMsodium chloride, 5 mM calcium chloride, pH 6.0) to get a final proteinconcentration of 1.0+/−0.25 mg/ml. Then the pH of the solution iscorrected to 6.0 by drop wise addition of a 0.5 N aqueous NaOH solution.Subsequently, a 40 mM aqueous sodium periodate solution is added within10 minutes to give a concentration of 50 μM. The oxidation reaction iscarried out for 30+/−5 min at a temperature (T) of T=+22+/−2° C. Thenthe reaction is stopped by addition of an aqueous L-cysteine solution (1M) within 15 minutes at T=+22+/−2° C. to give a final concentration of10 mM in the reaction mixture and incubation for 60+/−5 min.

The oxidized rFVIIa is further purified by anion exchange chromatographyon EMD TMAE (M) (Merck). The mixture is diluted with Buffer A (20 mMHepes, 5 mM CaCl₂, pH 6.5) to give a conductivity of 5 ms/cm. Thissolution is loaded onto the IEX column (bed height: 5.4 cm) with acolumn volume of 10 ml using a flow rate of 1.5 cm/min. This column issubsequently washed (flow rate: 1.5 cm/min) with 5 CV of a 92:8 mixture(w/w) of Buffer A and Buffer B (20 mM Hepes, 5 mM CaCl₂, 1.0 M NaCl, pH7.0). Then the oxidized rFVIIa is eluted with a 50:50 (w/w) mixture ofBuffer A and Buffer B followed by a postelution step with 5 CV of BufferB. The elution steps are carried out by use of a flow rate of 1.0cm/min.

Subsequently, the aminooxy-polysialic acid (PSA-ONH₂) reagent is addedin a 50-fold molar excess to the eluate containing the purified oxidizedrFVIIa within a maximum time period (t) of 15 minutes under gentlestirring. Then an aqueous m-toluidine solution (50 mM) is added within15 minutes to get a final concentration of 10 mM. The reaction mixtureis incubated for 120+/−10 min. in the dark at a temperature (T) ofT=+22+/−2° C. under gentle shaking.

The obtained PSA-rFVIIa conjugate is purified by Hydrophobic InteractionChromatography (HIC) using a Phenyl Sepharose FF low sub resin (GEHealthcare) packed into a column manufactured by GE Healthcare with abed height (h) of 15 cm and a resulting column volume (CV) of 81 ml.

The reaction mixture is spiked with ammonium acetate by addition of 50mM Hepes buffer, containing 350 mM sodium chloride, 8 M ammoniumacetate, 5 mM calcium chloride, pH 6.9. Two volumes of the reactionmixture are mixed with 1 volume of the ammonium acetate containingbuffer system and the pH value is corrected to pH 6.9 by drop wiseaddition of a 0.5 N aqueous NaOH solution. This mixture is loaded ontothe HIC column using a flow rate of 1 cm/min followed by a washing stepusing >3 CV equilibration buffer (50 mM Hepes, 350 mM sodium chloride,2.5 M ammonium acetate, 5 mM calcium chloride, pH 6.9).

For removal of reaction by-products and anti-chaotropic salt a secondwashing step is performed with >5CV washing buffer 1 (50 mM Hepes, 3 Msodium chloride, 5 mM calcium chloride, pH 6.9) in upflow mode at a flowrate of 2 cm/min. Then elution of purified rFVIIa conjugate is performedin down flow mode using a step gradient of 40% washing buffer 2 (50 mMHepes, 1.5 M sodium chloride, 5 mM calcium chloride, pH 6.9) and 60%elution buffer (20 mM Hepes, 5 mM calcium chloride, pH 7.5) at a flowrate of 1 cm/min. The elution of the PSA-rFVIIa conjugate is monitoredat UV 280 nm and the eluate containing the conjugate is collected within<4 CV. The post elution step is performed with >3 CV elution bufferunder the same conditions to separate minor and/or non modified rFVIIafrom the main product.

Finally, the purified conjugate is concentrated by ultra-/diafiltration(UF/DF) using a membrane made of regenerated cellulose with anappropriate molecular weight cut off (e.g. 10 kD MWCO, 88 cm²,Millipore).

The conjugate prepared by use of this procedure is analyticallycharacterized by measuring total protein, biological activity, anddetermination of the polysialyation degree by measuring the PSA content(resorcinol assay).

Method 2:

A starting weight or concentration of rFVIIa is dissolved in ortransferred to a reaction buffer (50 mM Hepes, 350 mM sodium chloride, 5mM calcium chloride, pH 6.0) to get a final protein concentration of1.0+/−0.25 mg/ml. Then the pH of the solution is corrected to 6.0 bydrop wise addition of a 0.5 N aqueous NaOH solution.

Subsequently, the aminooxy-polysialic acid (PSA-ONH₂) reagent is addedin a 50-fold molar excess to this rFVIIa solution within a maximum timeperiod (t) of 15 minutes under gentle stirring. Then an aqueousm-toluidine solution (50 mM) is added within 15 minutes to get a finalconcentration of 10 mM. Finally a 40 mM aqueous sodium periodatesolution is added to give a concentration of 150 μM.

The reaction mixture is incubated for 120+/−10 min. in the dark at atemperature (T) of T=+22+/−2° C. under gentle shaking. Then the reactionis stopped by the addition of an aqueous L-cysteine solution (1 M) togive a final concentration of 10 mM in the reaction mixture andincubation for 60+/−5 min.

The obtained PSA-rFVIIa conjugate is purified by Hydrophobic InteractionChromatography (HIC) using a Phenyl Sepharose FF low sub resin (GEHealthcare) packed into a column manufactured by GE Healthcare with abed height (h) of 15 cm and a resulting column volume (CV) of 81 ml.

The reaction mixture is spiked with ammonium acetate by addition of 50mM Hepes buffer, containing 350 mM sodium chloride, 8 M ammoniumacetate, 5 mM calcium chloride, pH 6.9. Two volumes of the reactionmixture is mixed with 1 volume of the ammonium acetate containing buffersystem and the pH value is corrected to pH 6.9 by drop wise addition ofan 0.5 N aqueous NaOH solution. This mixture is loaded onto the HICcolumn using a flow rate of 1 cm/min followed by a washing stepusing >3CV equilibration buffer (50 mM Hepes, 350 mM sodium chloride,2.5 M ammonium acetate, 5 mM calcium chloride, pH 6.9).

For removal of reaction by-products and anti-chaotropic salt a secondwashing step is performed with >5CV washing buffer 1 (50 mM Hepes, 3 Msodium chloride, 5 mM calcium chloride, pH 6.9) in upflow mode at a flowrate of 2 cm/min. Then elution of purified rFVIIa conjugate is performedin down flow mode using a step gradient of 40% washing buffer 2 (50 mMHepes, 1.5 M sodium chloride, 5 mM calcium chloride, pH 6.9) and 60%elution buffer (20 mM Hepes, 5 mM calcium chloride, pH 7.5) at a flowrate of 1 cm/min. The elution of the PSA-rFVIIa conjugate is monitoredat UV 280 nm and the eluate containing the conjugate was collectedwithin <4 CV. The post elution step is performed with >3 CV elutionbuffer under the same conditions to separate minor and/or non modifiedrFVIII from the main product.

Finally, the purified conjugate is concentrated by ultra-/diafiltration(UF/DF) using a membrane made of regenerated cellulose (Millipore).

The conjugates prepared by use of this procedure are analyticallycharacterized by measuring total protein, biological activity accordingto methods known in the art, and determination of the polysialyationdegree by measuring the PSA content (resorcinol assay).

Example 15 PEGylation of rFIX Using an Aminooxy-PEG Reagent andm-Toluidine as a Nucleophilic Catalyst

Method 1:

rFIX is PEGylated by use of a linear 20 kD PEGylation reagent containingan aminooxy group. An example of this type of reagent is the Sunbright®CA series from NOF (NOF Corp., Tokyo, Japan). A starting weight orconcentration of rFIX is dissolved in or transferred to a reactionbuffer (50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH6.0) to get a final protein concentration of 1.0+/−0.25 mg/ml. Then thepH of the solution is corrected to 6.0 by drop wise addition of a 0.5 Naqueous HCl solution. Subsequently, a 40 mM aqueous sodium periodatesolution is added within 10 minutes to give a concentration of 200 μM.The oxidation reaction is carried out for 30+/−5 min at a temperature(T) of T=+22+/−2° C. Then the reaction is stopped by addition of anaqueous L-cysteine solution (1 M) within 15 minutes at T=+22+/−2° C. togive a final concentration of 10 mM in the reaction mixture andincubation for 60+/−5 min.

The oxidized rFVIII is further purified by anion exchange chromatographyon EMD TMAE (M) (Merck). The mixture is diluted with Buffer A (20 mMHepes, 5 mM CaCl₂, pH 6.5) to give a conductivity of 5 mS/cm. Thissolution is loaded onto the IEX column (bed height: 5.4 cm) with acolumn volume of 10 ml using a flow rate of 1.5 cm/min. This column issubsequently washed (flow rate: 1.5 cm/min) with 5 CV of a 92:8 mixture(w/w) of Buffer A and Buffer B (20 mM Hepes, 5 mM CaCl₂, 1.0 M NaCl, pH7.0). Then the oxidized rFIX is eluted with a 50:50 (w/w) mixture ofBuffer A and Buffer B followed by a postelution step with 5 CV of BufferB. The elution steps are carried out by use of a flow rate of 1.0cm/min.

Subsequently, the aminooxy-PEG reagent with a MW of 20 kD reagent isadded in a 50-fold molar excess to the eluate containing the purifiedoxidized rFIX within a maximum time period (t) of 15 minutes undergentle stirring. Then an aqueous m-toluidine solution (50 mM) is addedwithin 15 minutes to get a final concentration of 10 mM. The reactionmixture is incubated for 120+/−10 min. in the dark at a temperature (T)of T=+22+/−2° C. under gentle shaking.

The obtained PEG-rFIX conjugate is purified by Hydrophobic InteractionChromatography (HIC) using a Phenyl Sepharose FF low sub resin (GEHealthcare) packed into a column manufactured by GE Healthcare with abed height (h) of 15 cm and a resulting column volume (CV) of 81 ml.

The reaction mixture is spiked with ammonium acetate by addition of 50mM Hepes buffer, containing 350 mM sodium chloride, 8 M ammoniumacetate, 5 mM calcium chloride, pH 6.9. Two volumes of the reactionmixture are mixed with 1 volume of the ammonium acetate containingbuffer system and the pH value is corrected to pH 6.9 by drop wiseaddition of a 0.5 N aqueous NaOH solution. This mixture is loaded ontothe HIC column using a flow rate of 1 cm/min followed by a washing stepusing >3 CV equilibration buffer (50 mM Hepes, 350 mM sodium chloride,2.5 M ammonium acetate, 5 mM calcium chloride, pH 6.9).

For removal of reaction by-products and anti-chaotropic salt a secondwashing step is performed with >5CV washing buffer 1 (50 mM Hepes, 3 Msodium chloride, 5 mM calcium chloride, pH 6.9) in upflow mode at a flowrate of 2 cm/min. Then elution of purified rFIX conjugate is performedin down flow mode using a step gradient of 40% washing buffer 2 (50 mMHepes, 1.5 M sodium chloride, 5 mM calcium chloride, pH 6.9) and 60%elution buffer (20 mM Hepes, 5 mM calcium chloride, pH 7.5) at a flowrate of 1 cm/min. The elution of the PEG-rFIX conjugate is monitored atUV 280 nm and the eluate containing the conjugate is collected within <4CV. The post elution step is performed with >3 CV elution buffer underthe same conditions to separate minor and/or non modified rFIX from themain product.

Finally, the purified conjugate is concentrated by ultra-/diafiltration(UF/DF) using a membrane made of regenerated cellulose with a molecularweight cut off 10 kD (88 cm2, Millipore).

The conjugate prepared by use of this procedure are analyticallycharacterized by measuring total protein and biological activityaccording to methods known in the art.

Method 2:

rFIX is PEGylated by use of a linear 20 kD PEGylation reagent containingan aminooxy group. An example of this type of reagent is the Sunbright®CA series from NOF (NOF Corp., Tokyo, Japan). An initial concentrationor weight of rFIX is transferred or dissolved in Hepes buffer (50 mMHepes, 150 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to get afinal protein concentration of 2 mg rFIX/ml. Subsequently, an 5 mMaqueous sodium periodate solution is added within 15 minutes to give afinal concentration of 100 μM, followed by addition of an 50 mM aqueousm-toluidine solution to get a final concentration of 10 mM within a timeperiod of 30 minutes. Then the aminooxy-PEG reagent with a MW of 20 kD(described above) is added to give a 20-fold molar reagent excess. Aftercorrection of the pH to 6.0 the mixture is incubated for 2 h in the darkat room temperature under gentle stirring and quenched for 15 min atroom temperature by the addition of a 1 M aqueous L-cysteine solution togive a final concentration of 10 mM.

The free rFIX is removed by means of ion exchange chromatography (IEC).The reaction mixture was diluted with appropriate amounts of Buffer A(50 mM Hepes, 5 mM CaCl₂, pH 7.5) to correct the solutions conductivityand pH value prior to load onto a 20 ml HiPrep QFF 16/10 column (GEHealthcare, Fairfield, Conn.) pre-equilibrated with Buffer A. Then thecolumn was eluted with Buffer B (50 mM Hepes, 1 M NaCl, 5 mM CaCl₂, pH7.5). Free rFIX was eluted by a step gradient using 25% of Buffer B,which results in a conductivity between 12-25 mS/cm in the obtainedfraction and the conjugate using a step gradient of 50% Buffer B, whichresults in a conductivity between 27-45 mS/cm in the conjugate fraction.The conductivity of the conjugate containing fraction is subsequentlyraised with Buffer C (50 mM Hepes, 5 M NaCl, 5 mM CaCl₂, pH 6.9; by useof anti-chaotropic salts e.g. ammonium acetate, etc) and loaded onto a20 ml HiPrep Butyl FF 16/10 column (GE Healthcare, Fairfield, Conn.; orcomparable HIC media) pre-equilibrated with Buffer D (50 mM Hepes, 3 MNaCl, 5 mM CaCl₂, pH 6.9). Free aminooxy-PEG reagent was washed outwithin 5 CV Buffer D. Subsequently, the conjugate was eluted with 100%Buffer E (50 mM Hepes, 5 mM CaCl₂, pH 7.4). The conjugate containingfractions are concentrated by UF/DF using a 10 kD membrane made ofregenerated cellulose (88 cm2, cut-off 10 kD, Millipore). The finaldiafiltration step is performed against Hepes buffer (50 mM Hepes, 5 mMCaCl₂, pH 7.5).

The preparation is analytically characterized by measuring total protein(Bradford and BCA procedure) and biological activity according to knownmethods.

Example 16 PEGylation of rFVIIa Using an Aminooxy-PEG Reagent andm-Toluidine as a Nucleophilic Catalyst

Method 1:

rFVIIa is PEGylated by use of a linear 20 kD PEGylation reagentcontaining an aminooxy group. An example of this type of reagent is theSunbright® CA series from NOF (NOF Corp., Tokyo, Japan). A startingweight or concentration of rFVIIa is dissolved in or transferred to areaction buffer (50 mM Hepes, 350 mM sodium chloride, 5 mM calciumchloride, pH 6.0) to get a final protein concentration of 1.0+/−0.25mg/ml. Then the pH of the solution is corrected to 6.0 by drop wiseaddition of a 0.5 N aqueous NaOH solution. Subsequently, a 40 mM aqueoussodium periodate solution is added within 10 minutes to give aconcentration of 50 μM. The oxidation reaction is carried out for 30+/−5min at a temperature (T) of T=+22+/−2° C. Then the reaction is stoppedby addition of an aqueous L-cysteine solution (1 M) within 15 minutes atT=+22+/−2° C. to give a final concentration of 10 mM in the reactionmixture and incubation for 60+/−5 min.

The oxidized rFVIIa is further purified by anion exchange chromatographyon EMD TMAE (M) (Merck). The mixture is diluted with Buffer A (20 mMHepes, 5 mM CaCl₂, pH 6.5) to give a conductivity of 5 mS/cm. Thissolution is loaded onto the IEX column (bed height: 5.4 cm) with acolumn volume of 10 ml using a flow rate of 1.5 cm/min. This column issubsequently washed (flow rate: 1.5 cm/min) with 5 CV of a 92:8 mixture(w/w) of Buffer A and Buffer B (20 mM Hepes, 5 mM CaCl₂, 1.0 M NaCl, pH7.0). Then the oxidized rFVIIa is eluted with a 50:50 (w/w) mixture ofBuffer A and Buffer B followed by a postelution step with 5 CV of BufferB. The elution steps are carried out by use of a flow rate of 1.0cm/min.

Subsequently, the aminooxy-PEG reagent with a MW of 20 kD reagent isadded in a 50-fold molar excess to the eluate containing the purifiedoxidized rFVIIa within a maximum time period (t) of 15 minutes undergentle stirring. Then an aqueous m-toluidine solution (50 mM) is addedwithin 15 minutes to get a final concentration of 10 mM. The reactionmixture is incubated for 120+/−10 min. in the dark at a temperature (T)of T=+22+/−2° C. under gentle shaking.

The obtained PEG-rFVIIa conjugate is purified by Hydrophobic InteractionChromatography (HIC) using a Phenyl Sepharose FF low sub resin (GEHealthcare) packed into a column manufactured by GE Healthcare with abed height (h) of 15 cm and a resulting column volume (CV) of 81 ml.

The reaction mixture is spiked with ammonium acetate by addition of 50mM Hepes buffer, containing 350 mM sodium chloride, 8 M ammoniumacetate, 5 mM calcium chloride, pH 6.9. Two volumes of the reactionmixture are mixed with 1 volume of the ammonium acetate containingbuffer system and the pH value is corrected to pH 6.9 by drop wiseaddition of a 0.5 N aqueous NaOH solution. This mixture is loaded ontothe HIC column using a flow rate of 1 cm/min followed by a washing stepusing >3 CV equilibration buffer (50 mM Hepes, 350 mM sodium chloride,2.5 M ammonium acetate, 5 mM calcium chloride, pH 6.9).

For removal of reaction by-products and anti-chaotropic salt a secondwashing step is performed with >5CV washing buffer 1 (50 mM Hepes, 3 Msodium chloride, 5 mM calcium chloride, pH 6.9) in upflow mode at a flowrate of 2 cm/min. Then elution of purified rFVIIa conjugate is performedin down flow mode using a step gradient of 40% washing buffer 2 (50 mMHepes, 1.5 M sodium chloride, 5 mM calcium chloride, pH 6.9) and 60%elution buffer (20 mM Hepes, 5 mM calcium chloride, pH 7.5) at a flowrate of 1 cm/min. The elution of the PEG-rFVIIa conjugate is monitoredat UV 280 nm and the eluate containing the conjugate is collected within<4 CV. The post elution step is performed with >3 CV elution bufferunder the same conditions to separate minor and/or non modified rFVIIafrom the main product.

Finally, the purified conjugate is concentrated by ultra-/diafiltration(UF/DF) using a membrane made of regenerated cellulose with a molecularweight cut off 10 kD (Millipore).

The conjugate prepared by use of this procedure are analyticallycharacterized by measuring total protein and biological activityaccording to methods known in the art.

Method 2:

rFVIIa is PEGylated by use of a linear 20 kD PEGylation reagentcontaining an aminooxy group. An example of this type of reagent is theSunbright® CA series from NOF (NOF Corp., Tokyo, Japan). An initialconcentration or weight of rFVIIa is transferred or dissolved in Hepesbuffer (50 mM Hepes, 150 mM sodium chloride, 5 mM calcium chloride, pH6.0) to get a final protein concentration of 2 mg rFVIIa/ml.Subsequently an 5 mM aqueous sodium periodate solution is added within15 minutes to give a final concentration of 100 μM, followed by additionof an 50 mM aqueous m-toluidine solution to get a final concentration of10 mM within a time period of 30 minutes. Then the aminooxy-PEG reagentwith a MW of 20 kD (described above) is added to give a 20-fold molarreagent excess. After correction of the pH to 6.0 the mixture isincubated for 2 h in the dark at room temperature under gentle stirringand quenched for 15 min at room temperature by the addition of a 1 Maqueous L-cysteine solution to give a final concentration of 10 mM.

The free rFVIIa is removed by means of ion exchange chromatography(IEC). The reaction mixture was diluted with appropriate amounts ofBuffer A (50 mM Hepes, 5 mM CaCl₂, pH 7.5) to correct the solutionsconductivity and pH value prior to load onto a 20 ml HiPrep QFF 16/10column (GE Healthcare, Fairfield, Conn.) pre-equilibrated with Buffer A.Then the column was eluted with Buffer B (50 mM Hepes, 1 M NaCl, 5 mMCaCl₂, pH 7.5). Free rFVIIa was eluted by a step gradient using 25% ofBuffer B, which results in a conductivity between 12-25 mS/cm in theobtained fraction and the conjugate using a step gradient of 50% BufferB, which results in a conductivity between 27-45 mS/cm in the conjugatefraction. The conductivity of the conjugate containing fraction issubsequently raised with Buffer C (50 mM Hepes, 5 M NaCl, 5 mM CaCl₂, pH6.9; by use of anti-chaotropic salts e.g. ammonium acetate) and loadedonto a 20 ml HiPrep Butyl FF 16/10 column (GE Healthcare, Fairfield,Conn.; or comparable HIC media) pre-equilibrated with Buffer D (50 mMHepes, 3 M NaCl, 5 mM CaCl₂, pH 6.9). Free PEG-reagent was washed outwithin 5 CV Buffer D. Subsequently the conjugate was eluted with 100%Buffer E (50 mM Hepes, 5 mM CaCl₂, pH 7.4). The conjugate containingfractions are concentrated by UF/DF using a 10 kD membrane made ofregenerated cellulose (88 cm2, cut-off 10 kD, Millipore). The finaldiafiltration step is performed against Hepes buffer (50 mM Hepes, 5 mMCaCl₂, pH 7.5).

The preparation is analytically characterized by measuring total protein(Bradford and BCA procedure) and biological activity according to knownmethods.

Example 17 Polysialylation of rFIX in the Presence of o-Amino BenzoicAcid

Method 1:

8.2 mg rFIX is dissolved in 4.0 ml histidine buffer, pH 6.0 (20 mML-histidine, 150 mM NaCl, 5 mM CaCl₂). Then 82 μl of an aqueous sodiumperiodate solution (5 mM) is added and the reaction mixture is incubatedfor 1 h in the dark at 4° C. under gentle stirring and quenched for 15min at room temperature by the addition of 4 μl of a 1 M aqueouscysteine solution. The mixture is subsequently subjected to UF/DFemploying Vivaspin 6 10 kD centrifugal filtrators to remove excessperiodate, quencher and the byproducts thereof.

The retentate (6.5 ml), containing oxidized rFIX, is mixed with 1.64 mlof an aqueous o-amino benzoic acid (50 mM) and incubated for 30 min atroom temperature. Then aminooxy-PSA reagent with a MW of 20 kD(described above) is added to give a 5-fold molar reagent excess. Thismixture was incubated for 2.5 h at room temperature in the dark undergentle stirring.

The further purification of the conjugate is carried out as describedherein.

Method 2:

A solution of 1 mg rFIX in 0.65 ml sodium phosphate buffer, pH 6.0containing a 5-fold molar excess of aminooxy-PSA reagent with a MW of 20kD (described above) was prepared. Then 333 μl of an aqueous o-aminobenzoic acid solution (30 mM) was added as nucleophilic catalyst to givea final concentration of 10 mM. Subsequently 20 μl of an aqueoussolution of NaIO₄ (5 mM) was added yielding in a final concentration of100 μM. The coupling process was performed for 2 hours in the dark undergentle shaking at room temperature and quenched for 15 min at roomtemperature by the addition of 1 μl of aqueous cysteine solution (1 M).The further purification of the conjugate is carried out as describedherein.

Example 18 Polysialylation of EPO Using Aminooxy-PSA and m-Toluidine asa Nucleophilic Catalyst

Method 1:

A starting concentration of erythropoietin (EPO) is transferred into areaction buffer (e.g. 50 mM Hepes, 350 mM sodium chloride, 5 mM calciumchloride, pH 6.0) and diluted to obtain a protein concentration of 1mg/ml. To this solution, NaIO₄ is added to give a final concentration of200 μM. The oxidation is carried at RT for 30 min in the dark undergentle shaking. The reaction is then quenched with cysteine (finalconcentration: 10 mM) for 60 min at RT.

The solution is next subjected to UF/DF employing Vivaspin centrifugalfiltrators to remove excess periodate, quencher and the byproductsthereof or, in the alternative, to an IEX column with a volume of 20 ml(Merck EMD TMAE (M)) which is equilibrated with Buffer A (20 mM Hepes, 5mM CaCl₂, pH 7.0). The column is equilibrated with 5 CV Buffer A. Theoxidized EPO is eluted with Buffer B (20 mM Hepes, 5 mM CaCl₂, 1M NaCl,pH 7.0). The EPO containing fractions are collected. The protein contentis determined (Coomassie, Bradford) and adjusted to 1 mg/ml withreaction buffer and adjusted to pH 6.0 by dropwise addition of 0.5M HCl.

A 50-fold molar excess of a aminooxy-PSA reagent with a MW of 20 kD(described above) is added followed by m-toluidine as a nucleophiliccatalyst (final concentration: 10 mM). The coupling reaction isperformed for 2 hours in the dark under gentle shaking at roomtemperature. The excess of aminooxy-PSA reagent is removed by means ofHIC. The conductivity of the reaction mixture is adjusted by adding abuffer containing ammonium acetate (50 mM Hepes, 350 mM sodium chloride,5 mM calcium chloride, 8 M ammonium acetate, pH 6.9) and loaded onto acolumn filled with 80 ml Phenyl Sepharose FF (GE Healthcare, Fairfield,Conn.) pre-equilibrated with 50 mM Hepes, 2.5 M ammonium acetate, 350 mMsodium chloride, 5 mM calcium chloride, pH 6.9. Subsequently, theconjugate is eluted with 50 mM Hepes buffer pH 7.5 containing 5 mMCaCl₂. Finally the PSA-EPO containing fractions are collected andsubjected to UF/DF by use of a membrane made of regenerated cellulose(MWCO 10 kD, 50 cm², Millipore). The preparation is next analyticallycharacterized by measuring total protein (Coomassie, Bradford) andbiological activity according to methods known in the art.

In an alternative embodiment, Method 1 is carried out as follows.

10 mg EPO is dissolved in 5 ml histidine buffer, pH 6.0 (20 mML-histidine, 150 mM NaCl). 100 μl of an aqueous sodium periodatesolution (5 mM) is then added and the reaction mixture is incubated for1 h in the dark at 4° C. under gentle stirring and quenched for 15 minat room temperature by the addition of 50 μl of a 1 M aqueous cysteinesolution. The mixture is subsequently subjected to UF/DF employingVivaspin 15R 10 kD centrifugal filtrators to remove excess periodate,quencher and the byproducts thereof.

The retentate (approx. 7 ml), containing oxidized EPO, is mixed with 2ml of an aqueous m-toluidine solution (50 mM) and incubated for 30 minat room temperature. Then aminooxy-PSA reagent with a MW of 20 kD(described above) is added to give a 5-fold molar reagent excess. Thismixture is incubated for 2.5 h at RT in the dark under gentle stirring.

The free EPO is removed by means of anion exchange chromatography (AEC).The reaction mixture is diluted with 20 ml Buffer A (50 mM Hepes, pH7.5) and loaded onto a 20 ml HiPrep QFF 16/10 column (GE Healthcare,Fairfield, Conn.) pre-equilibrated with Buffer A. Then the column iseluted with Buffer B (50 mM Hepes, 1 M NaCl, pH 7.5). Free EPO is elutedby washing the column with 25% Buffer B and the conjugate at 50% BufferB. The conductivity of the conjugate containing fractions issubsequently raised to ˜190 mS/cm with Buffer C (50 mM Hepes, 5 M NaCl,pH 6.9) and loaded onto a 20 ml HiPrep Butyl FF 16/10 column (GEHealthcare, Fairfield, Conn.) pre-equilibrated with Buffer D (50 mMHepes, 3 M NaCl, pH 6.9). Free PSA-reagent is washed out within 5 CVBuffer D. Subsequently, the conjugate is eluted with 100% Buffer E (50mM Hepes, pH 7.4). The conjugate containing fractions are concentratedby UF/DF using a 10 kD membrane made of regenerated cellulose (88 cm2,cut-off 10 kD/Millipore). The final diafiltration step is performedagainst histidine buffer, pH 7.2 containing 150 mM NaCl. The preparationis analytically characterized by measuring total protein (Bradford) andbiological activity according to methods known in the art. For thePSA-EPO conjugate a specific activity of >50% in comparison to nativeEPO is determined. The conjugate is additionally analyticallycharacterized by Size Exclusion HPLC using a Agilent 1200 HPLC systemequipped with a Shodex KW 803 column under conditions as previouslydescribed (Kolarich et al, Transfusion 2006; 46:1959-77). It is shownthat the preparation contains no free EPO.

Method 2:

EPO is transferred or dissolved in reaction buffer (e.g. 50 mM Hepes,350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to get a finalprotein concentration of 1.0+/−0.25 mg/ml. Then the pH of the solutionis corrected to 6.0 by drop wise addition of a 0.5 N aqueous HClsolution. Subsequently, a 40 mM aqueous sodium periodate solution isadded within 10 minutes to give a concentration of 200 μM. The oxidationreaction is carried out for 30+/−5 min at a temperature (T) ofT=+22+/−2° C. Then the reaction is stopped by addition of an aqueousL-cysteine solution (1 M) within 15 minutes at T=+22+/−2° C. to give afinal concentration of 10 mM in the reaction mixture and incubation for60+/−5 min.

The oxidized EPO is further purified by ion exchange chromatography. Theoxidized EPO containing fractions of the eluate are collected and usedfor the conjugation reaction.

The aminooxy-polysialic acid (PSA-ONH₂) reagent is added in a 50-foldmolar excess to the eluate containing the purified oxidized EPO within amaximum time period (t) of 15 minutes under gentle stirring. Then anaqueous m-toluidine solution (50 mM) is added within 15 minutes to get afinal concentration of 10 mM. The reaction mixture is incubated for120+/−10 min. at pH 6.0 in the dark at a temperature (T) of T=+22+/−2°C. under gentle shaking (protein concentration: 1 mg/ml).

The obtained PSA-EPO conjugate is further purified by ion exchangechromatography. The PSA-EPO conjugate containing fractions are collectedand concentrated by ultra-/diafiltration (UF/DF) using a membrane madeof regenerated cellulose with an appropriate molecular weight cut off(Millipore).

The conjugate prepared by use of this procedure is analyticallycharacterized by measuring total protein, biological activity, anddetermination of the polysialyation degree by measuring the PSA content(resorcinol assay).

Method 3:

Erythropoietin (EPO) is transferred into reaction buffer (50 mM Hepes,350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) and diluted toobtain a protein concentration of 1 mg/ml. A 50 fold molar excess of aaminooxy-PSA reagent with a MW of 20 kD (described above) is addedfollowed by m-toluidine as a nucleophilic catalyst (10 mM finalconcentration) and NaIO4 (final concentration: 400 μM). The couplingreaction is performed for 2 hours in the dark under gentle shaking atroom temperature. Subsequently, the reaction is quenched with cysteinefor 60 min at RT (cysteine concentration: 10 mM). Then the conductivityof the reaction mixture is adjusted by adding a buffer containingammonium acetate (50 mM Hepes, 350 mM sodium chloride, 5 mM calciumchloride, 8 M ammonium acetate, pH 6.9) and loaded onto a column filledwith Phenyl Sepharose FF (GE Healthcare, Fairfield, Conn.)pre-equilibrated with 50 mM Hepes, 2.5 M ammonium acetate, 350 mM sodiumchloride, 5 mM calcium chloride, 0.01% Tween 80, pH 6.9. Subsequently,the conjugate is eluted with 50 mM Hepes, 5 mM calcium chloride, pH 7.5.Finally, the PSA-EPO containing fractions are collected and subjected toUF/DF by use of a membrane made of regenerated cellulose (MWCO 10 kD, 88cm², Millipore). The preparation is analytically characterized bymeasuring total protein (Bradford) and biological activity according tomethods known in the art.

In an alternative embodiment, Method 3 is carried out as follows. 10 mgEPO is dissolved in 8 ml histidine buffer, pH 6.0 (20 mM L-histidine,150 mM NaCl). 200 μl of an aqueous sodium periodate solution (5 mM) and2 ml of an aqueous m-toluidine solution (50 mM) are then added.Subsequently, the aminooxy-PSA reagent with a MW of 20 kD (describedabove) is added to give a 5-fold molar reagent excess. The mixture isincubated for 2 h in the dark at room temperature under gentle stirringand quenched for 15 min at room temperature by the addition of 100 μl of1 M aqueous cysteine solution.

The free EPO is removed by means of anion exchange chromatography (AEC).The reaction mixture is diluted with 20 ml Buffer A (50 mM Hepes, pH7.5) and loaded onto a 20 ml HiPrep QFF 16/10 column (GE Healthcare,Fairfield, Conn.) pre-equilibrated with Buffer A. Then the column iseluted with Buffer B (50 mM Hepes, 1 M NaCl, pH 7.5). Free EPO is elutedby washing the column with 25% Buffer B and the conjugate at 50% BufferB. The conductivity of the conjugate containing fractions issubsequently raised to ˜190 mS/cm with Buffer C (50 mM Hepes, 5 M NaCl,pH 6.9) and loaded onto a 20 ml HiPrep Butyl FF 16/10 column (GEHealthcare, Fairfield, Conn.) pre-equilibrated with Buffer D (50 mMHepes, 3 M NaCl, pH 6.9). Free PSA-reagent is washed out within 5 CVBuffer D. Subsequently, the conjugate is eluted with 100% Buffer E (50mM Hepes, pH 7.4). The conjugate containing fractions are concentratedby UF/DF using a 10 kD membrane made of regenerated cellulose (88 cm2,cut-off 10 kD, Millipore). The final diafiltration step is performedagainst histidine buffer, pH 7.2 containing 150 mM NaCl. The preparationis analytically characterized by measuring total protein (Bradford) andbiological activity according to methods known in the art. For thePSA-EPO conjugate a specific activity of >50% in comparison to nativeEPO is determined. The conjugate is additionally analyticallycharacterized by Size Exclusion HPLC using a Agilent 1200 HPLC systemequipped with a Shodex KW 803 column under conditions as previouslydescribed (Kolarich et al, Transfusion 2006; 46:1959-77). It is shownthat the preparation contains no free EPO.

Method 4:

EPO is dissolved in or transferred to a reaction buffer (e.g. 50 mMHepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to get afinal protein concentration of 1.0+/−0.25 mg/ml. Then the pH of thesolution is corrected to 6.0 by drop wise addition of a 0.5 N aqueousHCl solution.

Subsequently, the aminooxy-polysialic acid (PSA-ONH₂) reagent is addedin a 50-fold molar excess to this EPO solution within a maximum timeperiod (t) of 15 minutes under gentle stirring. Then an aqueousm-toluidine solution (50 mM) is added within 15 minutes to get a finalconcentration of 10 mM. Finally a 40 mM aqueous sodium periodatesolution is added to give a concentration of 400 μM.

The reaction mixture is incubated for 120+/−10 min. in the dark at atemperature (T) of T=+22+/−2° C. under gentle shaking. Then the reactionis stopped by the addition of an aqueous L-cysteine solution (1 M) togive a final concentration of 10 mM in the reaction mixture andincubation for 60+/−5 min.

The obtained PSA-EPO conjugate is purified by ion-exchangechromatography. The PSA-EPO containing fractions of the eluate arecollected and concentrated by ultra-/diafiltration (UF/DF) using amembrane made of regenerated cellulose (MWCO 10 kD, 88 cm², Millipore).

The conjugates prepared by use of this procedure are analyticallycharacterized by measuring total protein, biological activity accordingto methods known in the art, and determination of the polysialyationdegree by measuring the PSA content (resorcinol assay).

Example 19 Polysialylation of Ang-2 Using Aminooxy-PSA and m-Toluidineas a Nucleophilic Catalyst

Method 1:

A starting concentration of angiopoietin-2 (Ang-2) is transferred into areaction buffer (e.g. 50 mM Hepes, 350 mM sodium chloride, 5 mM calciumchloride, pH 6.0) and diluted to obtain a protein concentration of 1mg/ml. To this solution, NaIO₄ is added to give a final concentration of200 μM. The oxidation is carried at RT for 30 min in the dark undergentle shaking. The reaction is then quenched with cysteine (finalconcentration: 10 mM) for 60 min at RT.

The solution is next subjected to UF/DF employing Vivaspin centrifugalfiltrators to remove excess periodate, quencher and the byproducts, or,in the alternative, subjected to an IEX column with a volume of 20 ml(Merck EMD TMAE (M)) which is equilibrated with Buffer A (20 mM Hepes, 5mM CaCl₂, pH 7.0). The column is equilibrated with 5 CV Buffer A. Theoxidized Ang-2 is eluted with Buffer B (20 mM Hepes, 5 mM CaCl₂, 1 MNaCl, pH 7.0). The Ang-2 containing fractions are collected. The proteincontent is determined (Coomassie, Bradford) and adjusted to 1 mg/ml withreaction buffer and adjusted to pH 6.0 by dropwise addition of 0.5 MHCl.

A 50-fold molar excess of aminooxy-PSA reagent with a MW of 20 kD(described above) is added followed by m-toluidine as a nucleophiliccatalyst (final concentration: 10 mM). The coupling reaction isperformed for 2 hours in the dark under gentle shaking at roomtemperature. The excess of aminooxy reagent is removed by means of HIC.The conductivity of the reaction mixture is adjusted by adding a buffercontaining ammonium acetate (50 mM Hepes, 350 mM sodium chloride, 5 mMcalcium chloride, 8 M ammonium acetate, pH 6.9) and loaded onto a columnfilled with 80 ml Phenyl Sepharose FF (GE Healthcare, Fairfield, Conn.)pre-equilibrated with 50 mM Hepes, 2.5 M ammonium acetate, 350 mM sodiumchloride, 5 mM calcium chloride, pH 6.9. Subsequently, the conjugate iseluted with 50 mM Hepes buffer pH 7.5 containing 5 mM CaCl₂. Finally,the PSA-Ang-2-containing fractions are collected and subjected to UF/DFby use of a membrane made of regenerated cellulose (Millipore). Thepreparation is next analytically characterized by measuring totalprotein (Coomassie, Bradford) and biological activity according tomethods known in the art.

In an alternative embodiment, Method 1 is carried out as follows.Angiopoietin-2 (Ang-2) is transferred into a reaction buffer (e.g., 50mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) anddiluted to obtain a protein concentration of 1 mg/ml. To this solution,NaIO₄ is added to give a final concentration of 200 μM. The oxidation iscarried at RT for 30 min in the dark under gentle shaking. The reactionis then quenched with cysteine (final concentration: 10 mM) for 60 minat R.T.

The solution is next subjected to UF/DF employing Vivaspin centrifugalfiltrators to remove excess periodate, quencher and the byproductsthereof.

A 50-fold molar excess of aminooxy-PSA reagent with a MW of 20 kD(described above) is added followed by m-toluidine as a nucleophiliccatalyst (final concentration: 10 mM). The coupling reaction isperformed for 2 hours in the dark under gentle shaking at roomtemperature. The excess of aminooxy reagent is removed by means of ionexchange chromatography. The PSA-Ang-2 conjugate-containing fractions ofthe eluate are collected and subjected to UF/DF by use of a membranemade of regenerated cellulose (Millipore). The preparation is nextanalytically characterized by measuring total protein (Coomassie,Bradford) and biological activity according to methods known in the art.

Method 2:

Ang-2 is transferred or dissolved in reaction buffer (e.g. 50 mM Hepes,350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to get a finalprotein concentration of 1.0+/−0.25 mg/ml. Then the pH of the solutionis corrected to 6.0 by drop wise addition of a 0.5 N aqueous HClsolution. Subsequently, a 40 mM aqueous sodium periodate solution isadded within 10 minutes to give a concentration of 200 μM. The oxidationreaction is carried out for 30+/−5 min at a temperature (T) ofT=+22+/−2° C. Then the reaction is stopped by addition of an aqueousL-cysteine solution (1 M) within 15 minutes at T=+22+/−2° C. to give afinal concentration of 10 mM in the reaction mixture and incubation for60+/−5 min.

The oxidized Ang-2 is further purified by ion exchange chromatography.The oxidized Ang-2 containing fractions of the eluate are collected andused for the conjugation reaction.

The aminooxy-polysialic acid (PSA-ONH₂) reagent is added in a 50-foldmolar excess to the eluate containing the purified oxidized Ang-2 withina maximum time period (t) of 15 minutes under gentle stirring. Then anaqueous m-toluidine solution (50 mM) is added within 15 minutes to get afinal concentration of 10 mM. The reaction mixture is incubated for120+/−10 min. at pH 6.0 in the dark at a temperature (T) of T=+22+/−2°C. under gentle shaking (protein concentration: 1 mg/ml).

The obtained PSA-Ang-2 conjugate is further purified by ion-exchangechromatography.

The PSA-Ang-2 conjugate containing fractions are collected andconcentrated by ultra-/diafiltration (UF/DF) using a membrane made ofregenerated cellulose with an appropriate molecular weight cut off(Millipore).

The conjugate prepared by use of this procedure is analyticallycharacterized by measuring total protein, biological activity, anddetermination of the polysialyation degree by measuring the PSA content(resorcinol assay).

Method 3:

Angiopoietin-2 (Ang-2) is transferred into reaction buffer (50 mM Hepes,350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) and diluted toobtain a protein concentration of 1 mg/ml. A 50 fold molar excess of aPSA aminooxy reagent with a MW of 20 kD (described above) is addedfollowed by m-toluidine as a nucleophilic catalyst (10 mM finalconcentration) and NaIO₄ (final concentration: 400 μM). The couplingreaction is performed for 2 hours in the dark under gentle shaking atroom temperature. Subsequently, the reaction is quenched with cysteinefor 60 min at RT (cysteine concentration: 10 mM). Then the conductivityof the reaction mixture is adjusted by adding a buffer containingammonium acetate (50 mM Hepes, 350 mM sodium chloride, 5 mM calciumchloride, 8 M ammonium acetate, pH 6.9) and loaded onto a column filledwith Phenyl Sepharose FF (GE Healthcare, Fairfield, Conn.)pre-equilibrated with 50 mM Hepes, 2.5 M ammonium acetate, 350 mM sodiumchloride, 5 mM calcium chloride, 0.01% Tween 80, pH 6.9. Subsequently,the conjugate is eluted with 50 mM Hepes, 5 mM calcium chloride, pH 7.5.Finally, the PSA Ang-2-containing fractions are collected and subjectedto UF/DF by use of a membrane made of regenerated cellulose (Millipore).The preparation is analytically characterized by measuring total protein(Bradford) and biological activity according to methods known in theart.

In an alternative embodiment, Method 3 is carried out as follows.Angiopoietin-2 (Ang-2) is transferred into reaction buffer (e.g. 50 mMHepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) anddiluted to obtain a protein concentration of 1 mg/ml. A 50-fold molarexcess of a PSA aminooxy reagent with a MW of 20 kD (described above) isadded followed by m-toluidine as a nucleophilic catalyst (10 mM finalconcentration) and NaIO₄ (final concentration: 400 μM). The couplingreaction is performed for 2 hours in the dark under gentle shaking atroom temperature. Subsequently, the reaction is quenched with cysteinefor 60 min at RT (cysteine concentration: 10 mM) and the conjugate ispurified by ion exchange chromatography. PSA Ang-2-containing fractionsof the eluate are collected and subjected to UF/DF by use of a membranemade of regenerated cellulose (Millipore). The preparation isanalytically characterized by measuring total protein (Bradford) andbiological activity according to methods known in the art.

Method 4:

Ang-2 is dissolved in or transferred to a reaction buffer (e.g. 50 mMHepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to get afinal protein concentration of 1.0+/−0.25 mg/ml. Then the pH of thesolution is corrected to 6.0 by drop wise addition of a 0.5 N aqueousHCl solution.

Subsequently, the aminooxy-polysialic acid (PSA-ONH₂) reagent is addedin a 50-fold molar excess to this Ang-2 solution within a maximum timeperiod (t) of 15 minutes under gentle stirring. Then an aqueousm-toluidine solution (50 mM) is added within 15 minutes to get a finalconcentration of 10 mM. Finally a 40 mM aqueous sodium periodatesolution is added to give a concentration of 400 μM.

The reaction mixture is incubated for 120+/−10 min. in the dark at atemperature (T) of T=+22+/−2° C. under gentle shaking. Then the reactionis stopped by the addition of an aqueous L-cysteine solution (1 M) togive a final concentration of 10 mM in the reaction mixture andincubation for 60+/−5 min.

The obtained PSA-Ang-2 conjugate is purified by ion-exchangechromatography. The PSA-Ang-2 containing fractions of the eluate arecollected and concentrated by ultra-/diafiltration (UF/DF) using amembrane made of regenerated cellulose (Millipore).

The conjugates prepared by use of this procedure are analyticallycharacterized by measuring total protein, biological activity accordingto methods known in the art, and determination of the polysialyationdegree by measuring the PSA content (resorcinol assay).

Example 20 Polysialylation of VEGF Using Aminooxy-PSA and m-Toluidine asa Nucleophilic Catalyst

Method 1:

A starting concentration of vascular endothelial growth factor (VEGF) istransferred into a reaction buffer (e.g., 50 mM Hepes, 350 mM sodiumchloride, 5 mM calcium chloride, pH 6.0) and diluted to obtain a proteinconcentration of 1 mg/ml. To this solution, NaIO₄ is added to give afinal concentration of 200 μM. The oxidation is carried at RT for 30 minin the dark under gentle shaking. The reaction is then quenched withcysteine (final concentration: 10 mM) for 60 min at RT.

The solution is next subjected to UF/DF employing Vivaspin centrifugalfiltrators to remove excess periodate, quencher and the byproductsthereof or, in the alternative, to an IEX column with a volume of 20 ml(Merck EMD TMAE (M)) which is equilibrated with Buffer A (20 mM Hepes, 5mM CaCl₂, pH 7.0). The column is equilibrated with 5 CV Buffer A. Theoxidized VEGF is eluted with Buffer B (20 mM Hepes, 5 mM CaCl₂, 1 MNaCl, pH 7.0). The VEGF containing fractions are collected. The proteincontent is determined (Coomassie, Bradford) and adjusted to 1 mg/ml withreaction buffer and adjusted to pH 6.0 by dropwise addition of 0.5MNaOH.

A 50-fold molar excess of aminooxy-PSA reagent with a MW of 20 kD(described above) is added followed by m-toluidine as a nucleophiliccatalyst (final concentration: 10 mM). The coupling reaction isperformed for 2 hours in the dark under gentle shaking at roomtemperature. The excess of aminooxy reagent is removed by means of HIC.The conductivity of the reaction mixture is adjusted by adding a buffercontaining ammonium acetate (50 mM Hepes, 350 mM sodium chloride, 5 mMcalcium chloride, 8 M ammonium acetate, pH 6.9) and loaded onto a columnfilled with 80 ml Phenyl Sepharose FF (GE Healthcare, Fairfield, Conn.)pre-equilibrated with 50 mM Hepes, 2.5 M ammonium acetate, 350 mM sodiumchloride, 5 mM calcium chloride, pH 6.9. Subsequently, the conjugate iseluted with 50 mM Hepes buffer pH 7.5 containing 5 mM CaCl₂. Finally thePSA-VEGF-containing fractions are collected and subjected to UF/DF byuse of a membrane made of regenerated cellulose (Millipore). Thepreparation is next analytically characterized by measuring totalprotein (Coomassie, Bradford) and biological activity according tomethods known in the art.

In an alternative embodiment, Method 1 is carried out as follows.Vascular endothelial growth factor (VEGF) is transferred into a reactionbuffer (e.g., 50 mM Hepes, 350 mM sodium chloride, 5 mM calciumchloride, pH 6.0) and diluted to obtain a protein concentration of 1mg/ml. To this solution, NaIO₄ is added to give a final concentration of200 μM. The oxidation is carried at RT for 30 min in the dark undergentle shaking. The reaction is then quenched with cysteine (finalconcentration: 10 mM) for 60 min at RT.

The solution is next subjected to UF/DF employing Vivaspin centrifugalfiltrators to remove excess periodate, quencher and the byproductsthereof.

A 50-fold molar excess of aminooxy-PSA reagent with a MW of 20 kD(described above) is added followed by m-toluidine as a nucleophiliccatalyst (final concentration: 10 mM). The coupling reaction isperformed for 2 hours in the dark under gentle shaking at roomtemperature. The excess of aminooxy reagent is removed by means of ionexchange chromatography. The PSA-VEGF-containing fractions of the eluateare collected and subjected to UF/DF by use of a membrane made ofregenerated cellulose (Millipore). The preparation is next analyticallycharacterized by measuring total protein (Coomassie, Bradford) andbiological activity according to methods known in the art.

Method 2:

VEGF is transferred or dissolved in reaction buffer (e.g. 50 mM Hepes,350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to get a finalprotein concentration of 1.0+/−0.25 mg/ml. Then the pH of the solutionis corrected to 6.0 by drop wise addition of a 0.5 N aqueous HClsolution. Subsequently a 40 mM aqueous sodium periodate solution isadded within 10 minutes to give a concentration of 200 μM. The oxidationreaction is carried out for 30+/−5 min at a temperature (T) ofT=+22+/−2° C. Then the reaction is stopped by addition of an aqueousL-cysteine solution (1 M) within 15 minutes at T=+22+/−2° C. to give afinal concentration of 10 mM in the reaction mixture and incubation for60+/−5 min.

The oxidized VEGF is further purified by ion exchange chromatography.The oxidized VEGF containing fractions of the eluate are collected andused for the conjugation reaction.

The aminooxy-polysialic acid (PSA-ONH₂) reagent is added in a 50-foldmolar excess to the eluate containing the purified oxidized VEGF withina maximum time period (t) of 15 minutes under gentle stirring. Then anaqueous m-toluidine solution (50 mM) is added within 15 minutes to get afinal concentration of 10 mM. The reaction mixture is incubated for120+/−10 min. at pH 6.0 in the dark at a temperature (T) of T=+22+/−2°C. under gentle shaking (protein concentration: 1 mg/ml).

The obtained PSA-VEGF conjugate is further purified by ion exchangechromatography. The PSA-VEGF conjugate containing fractions arecollected and concentrated by ultra-/diafiltration (UF/DF) using amembrane made of regenerated cellulose with an appropriate molecularweight cut off (Millipore).

The conjugate prepared by use of this procedure is analyticallycharacterized by measuring total protein, biological activity, anddetermination of the polysialyation degree by measuring the PSA content(resorcinol assay).

Method 3:

Vascular endothelial growth factor (VEGF) is transferred into reactionbuffer (50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH6.0) and diluted to obtain a protein concentration of 1 mg/ml. A 50-foldmolar excess of a PSA aminooxy reagent with a MW of 20 kD (describedabove) is added followed by m-toluidine as a nucleophilic catalyst (10mM final concentration) and NaIO₄ (final concentration: 400 μM). Thecoupling reaction is performed for 2 hours in the dark under gentleshaking at room temperature. Subsequently, the reaction is quenched withcysteine for 60 min at RT (cysteine concentration: 10 mM). Then theconductivity of the reaction mixture is adjusted by adding a buffercontaining ammonium acetate (50 mM Hepes, 350 mM sodium chloride, 5 mMcalcium chloride, 8 M ammonium acetate, pH 6.9) and loaded onto a columnfilled with Phenyl Sepharose FF (GE Healthcare, Fairfield, Conn.)pre-equilibrated with 50 mM Hepes, 2.5 M ammonium acetate, 350 mM sodiumchloride, 5 mM calcium chloride, 0.01% Tween 80, pH 6.9. Subsequentlythe conjugate is eluted with 50 mM Hepes, 5 mM calcium chloride, pH 7.5.Finally, the PSA-VEGF containing fractions are collected and subjectedto UF/DF by use of a membrane made of regenerated cellulose (Millipore).The preparation is analytically characterized by measuring total protein(Bradford) and biological activity according to methods known in theart.

In an alternative embodiment, Method 3 is carried out as follows.Vascular endothelial growth factor (VEGF) is transferred into reactionbuffer (e.g. 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride,pH 6.0) and diluted to obtain a protein concentration of 1 mg/ml. A50-fold molar excess of aminooxy-PSA reagent with a MW of 20 kD(described above) is added followed by m-toluidine as a nucleophiliccatalyst (10 mM final concentration) and NaIO₄ (final concentration: 400μM). The coupling reaction is performed for 2 hours in the dark undergentle shaking at room temperature. Subsequently, the reaction isquenched with cysteine for 60 min at RT (cysteine concentration: 10 mM)and the conjugate is purified by ion exchange chromatography. ThePSA-VEGF containing fractions of the eluate are collected and subjectedto UF/DF by use of a membrane made of regenerated cellulose (Millipore).The preparation is analytically characterized by measuring total protein(Bradford) and biological activity according to methods known in theart.

Method 4:

VEGF is dissolved in or transferred to a reaction buffer (e.g. 50 mMHepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to get afinal protein concentration of 1.0+/−0.25 mg/ml. Then the pH of thesolution is corrected to 6.0 by drop wise addition of a 0.5 N aqueousHCl solution.

Subsequently, the aminooxy-polysialic acid (PSA-ONH₂) reagent is addedin a 50-fold molar excess to this VEGF solution within a maximum timeperiod (t) of 15 minutes under gentle stirring. Then an aqueousm-toluidine solution (50 mM) is added within 15 minutes to get a finalconcentration of 10 mM. Finally a 40 mM aqueous sodium periodatesolution is added to give a concentration of 400 μM.

The reaction mixture is incubated for 120+/−10 min. in the dark at atemperature (T) of T=+22+/−2° C. under gentle shaking. Then the reactionis stopped by the addition of an aqueous L-cysteine solution (1 M) togive a final concentration of 10 mM in the reaction mixture andincubation for 60+/−5 min.

The obtained VEGF-conjugate is purified by ion-exchange chromatography.The PSA-VEGF containing fractions of the eluate are collected andconcentrated by ultra-/diafiltration (UF/DF) using a membrane made ofregenerated cellulose (Millipore).

The conjugates prepared by use of this procedure are analyticallycharacterized by measuring total protein, biological activity accordingto methods known in the art, and determination of the polysialyationdegree by measuring the PSA content (resorcinol assay).

Example 21 Polysialylation of EGF Using Aminooxy-PSA and m-Toluidine asa Nucleophilic Catalyst

Method 1:

A starting concentration of epidermal growth factor (EGF) is transferredinto a reaction buffer (e.g., 50 mM Hepes, 350 mM sodium chloride, 5 mMcalcium chloride, pH 6.0) and diluted to obtain a protein concentrationof 1 mg/ml. To this solution, NaIO₄ is added to give a finalconcentration of 200 μM. The oxidation is carried at RT for 30 min inthe dark under gentle shaking. The reaction is then quenched withcysteine (final concentration: 10 mM) for 60 min at R.T.

The solution is next subjected to UF/DF employing Vivaspin centrifugalfiltrators to remove excess periodate, quencher and the byproductsthereof or, in the alternative, to an IEX column with a volume of 20 ml(Merck EMD TMAE (M)) which is equilibrated with Buffer A (20 mM Hepes, 5mM CaCl₂, pH 7.0). The column is equilibrated with 5 CV Buffer A. Theoxidized EGF is eluted with Buffer B (20 mM Hepes, 5 mM CaCl₂, 1M NaCl,pH 7.0). The EGF containing fractions are collected. The protein contentis determined (Coomassie, Bradford) and adjusted to 1 mg/ml withreaction buffer and adjusted to pH 6.0 by dropwise addition of 0.5M HCl.

A 50-fold molar excess of aminooxy-PSA reagent with a MW of 20 kD(described above) is added followed by m-toluidine as a nucleophiliccatalyst (final concentration: 10 mM). The coupling reaction isperformed for 2 hours in the dark under gentle shaking at roomtemperature. The excess of aminooxy reagent is removed by means of HIC.The conductivity of the reaction mixture is adjusted by adding a buffercontaining ammonium acetate (50 mM Hepes, 350 mM sodium chloride, 5 mMcalcium chloride, 8 M ammonium acetate, pH 6.9) and loaded onto a columnfilled with 80 ml Phenyl Sepharose FF (GE Healthcare, Fairfield, Conn.)pre-equilibrated with 50 mM Hepes, 2.5 M ammonium acetate, 350 mM sodiumchloride, 5 mM calcium chloride, pH 6.9. Subsequently, the conjugate iseluted with 50 mM Hepes buffer pH 7.5 containing 5 mM CaCl₂. Finally,the PSA-EGF containing fractions are collected and subjected to UF/DF byuse of a membrane made of regenerated cellulose (Millipore). Thepreparation is next analytically characterized by measuring totalprotein (Coomassie, Bradford) and biological activity according tomethods known in the art.

In an alternative embodiment, Method 1 is carried out as follows.Epidermal growth factor (EGF) is transferred into a reaction buffer(e.g., 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH6.0) and diluted to obtain a protein concentration of 1 mg/ml. To thissolution, NaIO₄ is added to give a final concentration of 200 μM. Theoxidation is carried at RT for 30 min in the dark under gentle shaking.The reaction is then quenched with cysteine (final concentration: 10 mM)for 60 min at R.T.

The solution is next subjected to UF/DF employing Vivaspin centrifugalfiltrators to remove excess periodate, quencher and the byproductsthereof.

A 50-fold molar excess of aminooxy-PSA reagent with a MW of 20 kD(described above) is added followed by m-toluidine as a nucleophiliccatalyst (final concentration: 10 mM). The coupling reaction isperformed for 2 hours in the dark under gentle shaking at roomtemperature. The excess of aminooxy reagent is removed by means of ionexchange chromatography. The PSA-EGF containing fractions of the eluateare collected and subjected to UF/DF by use of a membrane made ofregenerated cellulose (Millipore). The preparation is next analyticallycharacterized by measuring total protein (Coomassie, Bradford) andbiological activity according to methods known in the art.

Method 2:

EGF is transferred or dissolved in reaction buffer (e.g. 50 mM Hepes,350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to get a finalprotein concentration of 1.0+/−0.25 mg/ml. Then the pH of the solutionis corrected to 6.0 by drop wise addition of a 0.5 N aqueous HClsolution. Subsequently, a 40 mM aqueous sodium periodate solution isadded within 10 minutes to give a concentration of 200 μM. The oxidationreaction is carried out for 30+/−5 min at a temperature (T) ofT=+22+/−2° C. Then the reaction is stopped by addition of an aqueousL-cysteine solution (1 M) within 15 minutes at T=+22+/−2° C. to give afinal concentration of 10 mM in the reaction mixture and incubation for60+/−5 min.

The oxidized EGF is further purified by ion exchange chromatography. Theoxidized EGF containing fractions of the eluate are collected and usedfor the conjugation reaction.

The aminooxy-polysialic acid (PSA-ONH₂) reagent is added in a 50-foldmolar excess to the eluate containing the purified oxidized EGF within amaximum time period (t) of 15 minutes under gentle stirring. Then anaqueous m-toluidine solution (50 mM) is added within 15 minutes to get afinal concentration of 10 mM. The reaction mixture is incubated for120+/−10 min. at pH 6.0 in the dark at a temperature (T) of T=+22+/−2°C. under gentle shaking (protein concentration: 1 mg/ml).

The obtained PSA-EGF conjugate is further purified by ion exchangechromatography. The PSA-EGF conjugate containing fractions are collectedand concentrated by ultra-/diafiltration (UF/DF) using a membrane madeof regenerated cellulose with an appropriate molecular weight cut off(Millipore).

The conjugate prepared by use of this procedure is analyticallycharacterized by measuring total protein, biological activity, anddetermination of the polysialyation degree by measuring the PSA content(resorcinol assay).

Method 3:

Epidermal growth factor (EGF) is transferred into reaction buffer (50 mMHepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) anddiluted to obtain a protein concentration of 1 mg/ml. A 50-fold molarexcess of a PSA aminooxy reagent with a MW of 20 kD (described above) isadded followed by m-toluidine as a nucleophilic catalyst (10 mM finalconcentration) and NaIO₄ (final concentration: 400 μM). The couplingreaction is performed for 2 hours in the dark under gentle shaking atroom temperature. Subsequently, the reaction is quenched with cysteinefor 60 min at RT (cysteine concentration: 10 mM). Then the conductivityof the reaction mixture is adjusted by adding a buffer containingammonium acetate (50 mM Hepes, 350 mM sodium chloride, 5 mM calciumchloride, 8 M ammonium acetate, pH 6.9) and loaded onto a column filledwith Phenyl Sepharose FF (GE Healthcare, Fairfield, Conn.)pre-equilibrated with 50 mM Hepes, 2.5 M ammonium acetate, 350 mM sodiumchloride, 5 mM calcium chloride, 0.01% Tween 80, pH 6.9. Subsequentlythe conjugate is eluted with 50 mM Hepes, 5 mM calcium chloride, pH 7.5.Finally the PSA-EGF containing fractions are collected and subjected toUF/DF by use of a membrane made of regenerated cellulose (Millipore).The preparation is analytically characterized by measuring total protein(Bradford) and biological activity according to methods known in theart.

In an alternative embodiment, Method 3 is carried out as follows.Epidermal growth factor (EGF) is transferred into reaction buffer (e.g.50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) anddiluted to obtain a protein concentration of 1 mg/ml. A 50-fold molarexcess of a PSA aminooxy reagent with a MW of 20 kD (described above) isadded followed by m-toluidine as a nucleophilic catalyst (10 mM finalconcentration) and NaIO₄ (final concentration: 400 μM). The couplingreaction is performed for 2 hours in the dark under gentle shaking atroom temperature. Subsequently, the reaction is quenched with cysteinefor 60 min at RT (cysteine concentration: 10 mM) and the conjugate ispurified by ion exchange chromatography. The conjugate containingfractions of the eluate are collected and subjected to UF/DF by use of amembrane made of regenerated cellulose (Millipore). The preparation isanalytically characterized by measuring total protein (Bradford) andbiological activity according to methods known in the art.

Method 4:

EGF is dissolved in or transferred to a reaction buffer (e.g. 50 mMHepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to get afinal protein concentration of 1.0+/−0.25 mg/ml. Then the pH of thesolution is corrected to 6.0 by drop wise addition of a 0.5 N aqueousHCl solution.

Subsequently the aminooxy-polysialic acid (PSA-ONH₂) reagent is added ina 50-fold molar excess to this EGF-solution within a maximum time period(t) of 15 minutes under gentle stirring. Then an aqueous m-toluidinesolution (50 mM) is added within 15 minutes to get a final concentrationof 10 mM. Finally a 40 mM aqueous sodium periodate solution is added togive a concentration of 400 μM.

The reaction mixture is incubated for 120+/−10 min. in the dark at atemperature (T) of T=+22+/−2° C. under gentle shaking. Then the reactionis stopped by the addition of an aqueous L-cysteine solution (1 M) togive a final concentration of 10 mM in the reaction mixture andincubation for 60+/−5 min.

The obtained EGF-conjugate is purified by ion-exchange chromatography.The PSA-EGF containing fractions of the eluate are collected andconcentrated by ultra-/diafiltration (UF/DF) using a membrane made ofregenerated cellulose (Millipore).

The conjugates prepared by use of this procedure are analyticallycharacterized by measuring total protein, biological activity accordingto methods known in the art, and determination of the polysialyationdegree by measuring the PSA content (resorcinol assay).

Example 22 Polysialylation of NGF Using Aminooxy-PSA and m-Toluidine asa Nucleophilic Catalyst

Method 1:

A starting concentration of nerve growth factor (NGF) is transferredinto a reaction buffer (e.g., 50 mM Hepes, 350 mM sodium chloride, 5 mMcalcium chloride, pH 6.0) and diluted to obtain a protein concentrationof 1 mg/ml. To this solution, NaIO₄ is added to give a finalconcentration of 200 μM. The oxidation is carried at RT for 30 min inthe dark under gentle shaking. The reaction is then quenched withcysteine (final concentration: 10 mM) for 60 min at RT.

The solution is next subjected to UF/DF employing Vivaspin centrifugalfiltrators to remove excess periodate, quencher and the byproductsthereof or, in the alternative, to an IEX column with a volume of 20 ml(Merck EMD TMAE (M)) which is equilibrated with Buffer A (20 mM Hepes, 5mM CaCl₂, pH 7.0). The column is equilibrated with 5 CV Buffer A. Theoxidized NGF is eluted with Buffer B (20 mM Hepes, 5 mM CaCl₂, 1M NaCl,pH 7.0). The NGF containing fractions are collected. The protein contentis determined (Coomassie, Bradford) and adjusted to 1 mg/ml withreaction buffer and adjusted to pH 6.0 by dropwise addition of 0.5M HCl.

A 50-fold molar excess of aminooxy-PSA reagent with a MW of 20 kD(described above) is added followed by m-toluidine as a nucleophiliccatalyst (final concentration: 10 mM). The coupling reaction isperformed for 2 hours in the dark under gentle shaking at roomtemperature. The excess of aminooxy reagent is removed by means of HIC.The conductivity of the reaction mixture is adjusted by adding a buffercontaining ammonium acetate (50 mM Hepes, 350 mM sodium chloride, 5 mMcalcium chloride, 8 M ammonium acetate, pH 6.9) and loaded onto a columnfilled with 80 ml Phenyl Sepharose FF (GE Healthcare, Fairfield, Conn.)pre-equilibrated with 50 mM Hepes, 2.5 M ammonium acetate, 350 mM sodiumchloride, 5 mM calcium chloride, pH 6.9. Subsequently, the conjugate iseluted with 50 mM Hepes buffer pH 7.5 containing 5 mM CaCl₂. Finally,the PSA-NGF containing fractions are collected and subjected to UF/DF byuse of a membrane made of regenerated cellulose (Millipore). Thepreparation is next analytically characterized by measuring totalprotein (Coomassie, Bradford) and biological activity according tomethods known in the art.

In an alternative embodiment, Method 1 is carried out as follows. Nervegrowth factor (NGF) is transferred into a reaction buffer (e.g., 50 mMHepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) anddiluted to obtain a protein concentration of 1 mg/ml. To this solution,NaIO₄ is added to give a final concentration of 200 μM. The oxidation iscarried at RT for 30 min in the dark under gentle shaking. The reactionis then quenched with cysteine (final concentration: 10 mM) for 60 minat RT.

The solution is next subjected to UF/DF employing Vivaspin centrifugalfiltrators to remove excess periodate, quencher and the byproductsthereof.

A 50-fold molar excess of aminooxy-PSA reagent with a MW of 20 kD(described above) is added followed by m-toluidine as a nucleophiliccatalyst (final concentration: 10 mM). The coupling reaction isperformed for 2 hours in the dark under gentle shaking at roomtemperature. The excess of aminooxy reagent is removed by means of ionexchange chromatography. The PSA-NGF containing fractions of the eluateare collected and subjected to UF/DF by use of a membrane made ofregenerated cellulose (Millipore). The preparation is next analyticallycharacterized by measuring total protein (Coomassie, Bradford) andbiological activity according to methods known in the art.

Method 2:

NGF is transferred or dissolved in reaction buffer (e.g. 50 mM Hepes,350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to get a finalprotein concentration of 1.0+/−0.25 mg/ml. Then the pH of the solutionis corrected to 6.0 by drop wise addition of a 0.5 N aqueous HClsolution. Subsequently, a 40 mM aqueous sodium periodate solution isadded within 10 minutes to give a concentration of 200 μM. The oxidationreaction is carried out for 30+/−5 min at a temperature (T) ofT=+22+/−2° C. Then the reaction is stopped by addition of an aqueousL-cysteine solution (1 M) within 15 minutes at T=+22+/−2° C. to give afinal concentration of 10 mM in the reaction mixture and incubation for60+/−5 min.

The oxidized NGF is further purified by ion exchange chromatography. Theoxidized NGF containing fractions of the eluate are collected and usedfor the conjugation reaction.

The aminooxy-polysialic acid (PSA-ONH₂) reagent is added in a 50-foldmolar excess to the eluate containing the purified oxidized NGF within amaximum time period (t) of 15 minutes under gentle stirring. Then anaqueous m-toluidine solution (50 mM) is added within 15 minutes to get afinal concentration of 10 mM. The reaction mixture is incubated for120+/−10 min. at pH 6.0 in the dark at a temperature (T) of T=+22+/−2°C. under gentle shaking (protein concentration: 1 mg/ml).

The obtained PSA-NGF conjugate is further purified by ion exchangechromatography. The PSA-NGF conjugate containing fractions are collectedand concentrated by ultra-/diafiltration (UF/DF) using a membrane madeof regenerated cellulose with an appropriate molecular weight cut off(Millipore).

The conjugate prepared by use of this procedure is analyticallycharacterized by measuring total protein, biological activity, anddetermination of the polysialyation degree by measuring the PSA content(resorcinol assay).

Method 3:

Nerve growth factor (NGF) is transferred into reaction buffer (50 mMHepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) anddiluted to obtain a protein concentration of 1 mg/ml. A 50-fold molarexcess of aminooxy-PSA reagent with a MW of 20 kD (described above) isadded followed by m-toluidine as a nucleophilic catalyst (10 mM finalconcentration) and NaIO₄ (final concentration: 400 μM). The couplingreaction is performed for 2 hours in the dark under gentle shaking atroom temperature. Subsequently, the reaction is quenched with cysteinefor 60 min at RT (cysteine concentration: 10 mM). Then the conductivityof the reaction mixture is adjusted by adding a buffer containingammonium acetate (50 mM Hepes, 350 mM sodium chloride, 5 mM calciumchloride, 8 M ammonium acetate, pH 6.9) and loaded onto a column filledwith Phenyl Sepharose FF (GE Healthcare, Fairfield, Conn.)pre-equilibrated with 50 mM Hepes, 2.5 M ammonium acetate, 350 mM sodiumchloride, 5 mM calcium chloride, 0.01% Tween 80, pH 6.9. Subsequentlythe conjugate is eluted with 50 mM Hepes, 5 mM calcium chloride, pH 7.5.Finally, the PSA NGF-containing fractions are collected and subjected toUF/DF by use of a membrane made of regenerated cellulose (Millipore).The preparation is analytically characterized by measuring total protein(Bradford) and biological activity according to methods known in theart.

In an alternative embodiment, Method 3 is carried out as follows. Nervegrowth factor (NGF) is transferred into reaction buffer (e.g. 50 mMHepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) anddiluted to obtain a protein concentration of 1 mg/ml. A 50-fold molarexcess of aminooxy-PSA reagent with a MW of 20 kD (described above) isadded followed by m-toluidine as a nucleophilic catalyst (10 mM finalconcentration) and NaIO₄ (final concentration: 400 μM). The couplingreaction is performed for 2 hours in the dark under gentle shaking atroom temperature. Subsequently, the reaction is quenched with cysteinefor 60 min at RT (cysteine concentration: 10 mM) and the conjugate ispurified by ion exchange chromatography. Then the PSA-NGF containingfractions of the eluate are collected and subjected to UF/DF by use of amembrane made of regenerated cellulose (Millipore). The preparation isanalytically characterized by measuring total protein (Bradford) andbiological activity according to methods known in the art.

Method 4:

NGF is dissolved in or transferred to a reaction buffer (e.g. 50 mMHepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to get afinal protein concentration of 1.0+/−0.25 mg/ml. Then the pH of thesolution is corrected to 6.0 by drop wise addition of a 0.5 N aqueousHCl solution.

Subsequently, the aminooxy-polysialic acid (PSA-ONH₂) reagent is addedin a 50-fold molar excess to this NGF-solution within a maximum timeperiod (t) of 15 minutes under gentle stirring. Then an aqueousm-toluidine solution (50 mM) is added within 15 minutes to get a finalconcentration of 10 mM. Finally a 40 mM aqueous sodium periodatesolution is added to give a concentration of 400 μM.

The reaction mixture is incubated for 120+/−10 min. in the dark at atemperature (T) of T=+22+/−2° C. under gentle shaking. Then the reactionis stopped by the addition of an aqueous L-cysteine solution (1 M) togive a final concentration of 10 mM in the reaction mixture andincubation for 60+/−5 min.

The obtained NGF-conjugate is purified by ion-exchange chromatography.The PSA-NGF containing fractions of the eluate are collected andconcentrated by ultra-/diafiltration (UF/DF) using a membrane made ofregenerated cellulose (Millipore).

The conjugates prepared by use of this procedure are analyticallycharacterized by measuring total protein, biological activity accordingto methods known in the art, and determination of the polysialyationdegree by measuring the PSA content (resorcinol assay).

Example 23 Polysialylation of HGH Using Aminooxy-PSA and m-Toluidine asa Nucleophilic Catalyst

Method 1:

As described herein, the amino acid sequence of human growth hormone(HGH) is first modified to incorporate at least one glycosylation site.Following purification, HGH is glycosylated in vitro according tomethods known in the art.

A starting concentration of human growth hormone (HGH) is transferredinto a reaction buffer (e.g., 50 mM Hepes, 350 mM sodium chloride, 5 mMcalcium chloride, pH 6.0) and diluted to obtain a protein concentrationof 1 mg/ml. To this solution, NaIO₄ is added to give a finalconcentration of 200 μM. The oxidation is carried at RT for 30 min inthe dark under gentle shaking. The reaction is then quenched withcysteine (final concentration: 10 mM) for 60 min at RT.

The solution is next subjected to UF/DF employing Vivaspin centrifugalfiltrators to remove excess periodate, quencher and the byproductsthereof or, in the alternative, to an IEX column with a volume of 20 ml(Merck EMD TMAE (M)) which is equilibrated with Buffer A (20 mM Hepes, 5mM CaCl₂, pH 7.0). The column is equilibrated with 5 CV Buffer A. Theoxidized HGH is eluted with Buffer B (20 mM Hepes, 5 mM CaCl₂, 1 M NaCl,pH 7.0). The HGH containing fractions are collected. The protein contentis determined (Coomassie, Bradford) and adjusted to 1 mg/ml withreaction buffer and adjusted to pH 6.0 by dropwise addition of 0.5 MHCl.

A 50-fold molar excess of aminooxy-PSA reagent with a MW of 20 kD(described above) is added followed by m-toluidine as a nucleophiliccatalyst (final concentration: 10 mM). The coupling reaction isperformed for 2 hours in the dark under gentle shaking at roomtemperature. The excess of aminooxy reagent is removed by means of HIC.The conductivity of the reaction mixture is adjusted by adding a buffercontaining ammonium acetate (50 mM Hepes, 350 mM sodium chloride, 5 mMcalcium chloride, 8 M ammonium acetate, pH 6.9) and loaded onto a columnfilled with 80 ml Phenyl Sepharose FF (GE Healthcare, Fairfield, Conn.)pre-equilibrated with 50 mM Hepes, 2.5 M ammonium acetate, 350 mM sodiumchloride, 5 mM calcium chloride, pH 6.9. Subsequently the conjugate iseluted with 50 mM Hepes buffer pH 7.5 containing 5 mM CaCl₂. Finally,the PSA-HGH containing fractions are collected and subjected to UF/DF byuse of a membrane made of regenerated cellulose (Millipore). Thepreparation is next analytically characterized by measuring totalprotein (Coomassie, Bradford) and biological activity according tomethods known in the art.

In an alternative embodiment, Method 1 is carried out as follows. Asdescribed herein, the amino acid sequence of human growth hormone (HGH)is first modified to incorporate at least one glycosylation site.Following purification, HGH is glycosylated in vitro according tomethods known in the art. HGH is transferred into a reaction buffer(e.g., 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH6.0) and diluted to obtain a protein concentration of 1 mg/ml. To thissolution, NaIO₄ is added to give a final concentration of 200 μM. Theoxidation is carried at RT for 30 min in the dark under gentle shaking.The reaction is then quenched with cysteine (final concentration: 10 mM)for 60 min at RT.

The solution is next subjected to UF/DF employing Vivaspin centrifugalfiltrators to remove excess periodate, quencher and the byproductsthereof.

A 50-fold molar excess of aminooxy-PSA reagent with a MW of 20 kD(described above) is added followed by m-toluidine as a nucleophiliccatalyst (final concentration: 10 mM). The coupling reaction isperformed for 2 hours in the dark under gentle shaking at roomtemperature. The excess of aminooxy reagent is removed by means of ionexchange chromatography. The PSA-HGH containing fractions of the eluateare collected and subjected to UF/DF by use of a membrane made ofregenerated cellulose (Millipore). The preparation is next analyticallycharacterized by measuring total protein (Coomassie, Bradford) andbiological activity according to methods known in the art.

Method 2:

As described herein, the amino acid sequence of human growth hormone(HGH) is first modified to incorporate at least one glycosylation site.Following purification, HGH is glycosylated in vitro according tomethods known in the art.

HGH is transferred or dissolved in reaction buffer (e.g. 50 mM Hepes,350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to get a finalprotein concentration of 1.0+/−0.25 mg/ml. Then the pH of the solutionis corrected to 6.0 by drop wise addition of a 0.5 N aqueous HClsolution. Subsequently, a 40 mM aqueous sodium periodate solution isadded within 10 minutes to give a concentration of 200 μM. The oxidationreaction is carried out for 30+/−5 min at a temperature (T) ofT=+22+/−2° C. Then the reaction is stopped by addition of an aqueousL-cysteine solution (1 M) within 15 minutes at T=+22+/−2° C. to give afinal concentration of 10 mM in the reaction mixture and incubation for60+/−5 min.

The oxidized HGH is further purified by ion exchange chromatography. Theoxidized HGH containing fractions of the eluate are collected and usedfor the conjugation reaction.

The aminooxy-polysialic acid (PSA-ONH₂) reagent is added in a 50-foldmolar excess to the eluate containing the purified oxidized HGH within amaximum time period (t) of 15 minutes under gentle stirring. Then anaqueous m-toluidine solution (50 mM) is added within 15 minutes to get afinal concentration of 10 mM. The reaction mixture is incubated for120+/−10 min. at pH 6.0 in the dark at a temperature (T) of T=+22+/−2°C. under gentle shaking (protein concentration: 1 mg/ml).

The obtained PSA-HGH conjugate is further purified by ion exchangechromatography. The PSA-HGH conjugate containing fractions are collectedand concentrated by ultra-/diafiltration (UF/DF) using a membrane madeof regenerated cellulose with an appropriate molecular weight cut off(Millipore).

The conjugate prepared by use of this procedure is analyticallycharacterized by measuring total protein, biological activity, anddetermination of the polysialyation degree by measuring the PSA content(resorcinol assay).

Method 3:

As described herein, the amino acid sequence of human growth hormone(HGH) is first modified to incorporate at least one glycosylation site.Following purification, HGH is glycosylated in vitro according tomethods known in the art.

Human growth hormone (HGH) is transferred into reaction buffer (50 mMHepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) anddiluted to obtain a protein concentration of 1 mg/ml. A 50 fold molarexcess of aminooxy-PSA reagent with a MW of 20 kD (described above) isadded followed by m-toluidine as a nucleophilic catalyst (10 mM finalconcentration) and NaIO₄ (final concentration: 400 μM). The couplingreaction is performed for 2 hours in the dark under gentle shaking atroom temperature. Subsequently, the reaction is quenched with cysteinefor 60 min at RT (cysteine concentration: 10 mM). Then the conductivityof the reaction mixture is adjusted by adding a buffer containingammonium acetate (50 mM Hepes, 350 mM sodium chloride, 5 mM calciumchloride, 8 M ammonium acetate, pH 6.9) and loaded onto a column filledwith Phenyl Sepharose FF (GE Healthcare, Fairfield, Conn.)pre-equilibrated with 50 mM Hepes, 2.5 M ammonium acetate, 350 mM sodiumchloride, 5 mM calcium chloride, 0.01% Tween 80, pH 6.9. Subsequentlythe conjugate is eluted with 50 mM Hepes, 5 mM calcium chloride, pH 7.5.Finally, the PSA HGH-containing fractions are collected and subjected toUF/DF by use of a membrane made of regenerated cellulose (Millipore).The preparation is analytically characterized by measuring total protein(Bradford) and biological activity according to methods known in theart.

In an alternative embodiment, Method 3 is carried out as follows. Asdescribed herein, the amino acid sequence of human growth hormone (HGH)is first modified to incorporate at least one glycosylation site.Following purification, HGH is glycosylated in vitro according tomethods known in the art. HGH is transferred into reaction buffer (e.g.50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) anddiluted to obtain a protein concentration of 1 mg/ml. A 50 fold molarexcess of aminooxy-PSA reagent with a MW of 20 kD (described above) isadded followed by m-toluidine as a nucleophilic catalyst (10 mM finalconcentration) and NaIO₄ (final concentration: 400 μM). The couplingreaction is performed for 2 hours in the dark under gentle shaking atroom temperature. Subsequently, the reaction is quenched with cysteinefor 60 min at RT (cysteine concentration: 10 mM) and the conjugate ispurified by ion exchange chromatography. Then the PSA-HGH-containingfractions of the eluate are collected and subjected to UF/DF by use of amembrane made of regenerated cellulose (Millipore). The preparation isanalytically characterized by measuring total protein (Bradford) andbiological activity according to methods known in the art.

Method 4:

As described herein, the amino acid sequence of human growth hormone(HGH) is first modified to incorporate at least one glycosylation site.Following purification, HGH is glycosylated in vitro according tomethods known in the art.

HGH is dissolved in or transferred to a reaction buffer (e.g. 50 mMHepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to get afinal protein concentration of 1.0+/−0.25 mg/ml. Then the pH of thesolution is corrected to 6.0 by drop wise addition of a 0.5 N aqueousHCl solution.

Subsequently, the aminooxy-polysialic acid (PSA-ONH₂) reagent is addedin a 50-fold molar excess to this HGH-solution within a maximum timeperiod (t) of 15 minutes under gentle stirring. Then an aqueousm-toluidine solution (50 mM) is added within 15 minutes to get a finalconcentration of 10 mM. Finally a 40 mM aqueous sodium periodatesolution is added to give a concentration of 400 μM.

The reaction mixture is incubated for 120+/−10 min. in the dark at atemperature (T) of T=+22+/−2° C. under gentle shaking. Then the reactionis stopped by the addition of an aqueous L-cysteine solution (1 M) togive a final concentration of 10 mM in the reaction mixture andincubation for 60+/−5 min.

The obtained HGH-conjugate is purified by ion-exchange chromatography.The PSA-HGH containing fractions of the eluate are collected andconcentrated by ultra-/diafiltration (UF/DF) using a membrane made ofregenerated cellulose (Millipore).

The conjugates prepared by use of this procedure are analyticallycharacterized by measuring total protein, biological activity accordingto methods known in the art, and determination of the polysialyationdegree by measuring the PSA content (resorcinol assay).

Example 24

Polysialylation of TNF-alpha using aminooxy-PSA and m-toluidine as anucleophilic catalyst A starting concentration of tumor necrosisfactor-alpha (TNF-alpha) is transferred into a reaction buffer (e.g., 50mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) anddiluted to obtain a protein concentration of 1 mg/ml. To this solution,NaIO₄ is added to give a final concentration of 200 μM. The oxidation iscarried at RT for 30 min in the dark under gentle shaking. The reactionis then quenched with cysteine (final concentration: 10 mM) for 60 minat RT.

The solution is next subjected to UF/DF employing Vivaspin centrifugalfiltrators to remove excess periodate, quencher and the byproductsthereof or, in the alternative, to an IEX column with a volume of 20 ml(Merck EMD TMAE (M)) which is equilibrated with Buffer A (20 mM Hepes, 5mM CaCl₂, pH 7.0). The column is equilibrated with 5 CV Buffer A. Theoxidized TNF-alpha is eluted with Buffer B (20 mM Hepes, 5 mM CaCl₂, 1MNaCl, pH 7.0). The TNF-alpha containing fractions are collected. Theprotein content is determined (Coomassie, Bradford) and adjusted to 1mg/ml with reaction buffer and adjusted to pH 6.0 by dropwise additionof 0.5 M HCl.

A 50-fold molar excess of aminooxy-PSA reagent with a MW of 20 kD(described above) is added followed by m-toluidine as a nucleophiliccatalyst (final concentration: 10 mM). The coupling reaction isperformed for 2 hours in the dark under gentle shaking at roomtemperature. The excess of aminooxy reagent is removed by means of HIC.The conductivity of the reaction mixture is adjusted by adding a buffercontaining ammonium acetate (50 mM Hepes, 350 mM sodium chloride, 5 mMcalcium chloride, 8 M ammonium acetate, pH 6.9) and loaded onto a columnfilled with 80 ml Phenyl Sepharose FF (GE Healthcare, Fairfield, Conn.)pre-equilibrated with 50 mM Hepes, 2.5 M ammonium acetate, 350 mM sodiumchloride, 5 mM calcium chloride, pH 6.9. Subsequently, the conjugate iseluted with 50 mM Hepes buffer pH 7.5 containing 5 mM CaCl₂. Finally thePSA-TNF-alpha-containing fractions are collected and subjected to UF/DFby use of a membrane made of regenerated cellulose (Millipore). Thepreparation is next analytically characterized by measuring totalprotein (Coomassie, Bradford) and biological activity according tomethods known in the art.

In an alternative embodiment, Method 1 is carried out as follows. Tumornecrosis factor-alpha (TNF-alpha) is transferred into a reaction buffer(e.g. 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH6.0) and diluted to obtain a protein concentration of 1 mg/ml. To thissolution, NaIO₄ is added to give a final concentration of 200 μM. Theoxidation is carried at RT for 30 min in the dark under gentle shaking.The reaction is then quenched with cysteine (final concentration: 10 mM)for 60 min at RT.

The solution is next subjected to UF/DF employing Vivaspin centrifugalfiltrators to remove excess periodate, quencher and the byproductsthereof. A 50-fold molar excess of aminooxy-PSA reagent with a MW of 20kD (described above) is added followed by m-toluidine as a nucleophiliccatalyst (final concentration: 10 mM). The coupling reaction isperformed for 2 hours in the dark under gentle shaking at roomtemperature. The excess of aminooxy reagent is removed by means of ionexchange chromatography. The PSA-TNF-alpha containing fractions of theeluate are collected and subjected to UF/DF by use of a membrane made ofregenerated cellulose (Millipore). The preparation is next analyticallycharacterized by measuring total protein (Coomassie, Bradford) andbiological activity according to methods known in the art.

Method 2:

TNF-alpha is transferred or dissolved in reaction buffer (e.g. 50 mMHepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to get afinal protein concentration of 1.0+/−0.25 mg/ml. Then the pH of thesolution is corrected to 6.0 by drop wise addition of a 0.5 N aqueousHCl solution. Subsequently, a 40 mM aqueous sodium periodate solution isadded within 10 minutes to give a concentration of 200 μM. The oxidationreaction is carried out for 30+/−5 min at a temperature (T) ofT=+22+/−2° C. Then the reaction is stopped by addition of an aqueousL-cysteine solution (1 M) within 15 minutes at T=+22+/−2° C. to give afinal concentration of 10 mM in the reaction mixture and incubation for60+/−5 min.

The oxidized TNF-alpha is further purified by ion exchangechromatography. The oxidized TNF-alpha containing fractions of theeluate are collected and used for the conjugation reaction.

The aminooxy-polysialic acid (PSA-ONH₂) reagent is added in a 50-foldmolar excess to the eluate containing the purified oxidized TNF-alphawithin a maximum time period (t) of 15 minutes under gentle stirring.Then an aqueous m-toluidine solution (50 mM) is added within 15 minutesto get a final concentration of 10 mM. The reaction mixture is incubatedfor 120+/−10 min. at pH 6.0 in the dark at a temperature (T) ofT=+22+/−2° C. under gentle shaking (protein concentration: 1 mg/ml).

The obtained PSA-TNF-alpha conjugate is further purified by ion exchangechromatography. The PSA-TNF-alpha conjugate containing fractions arecollected and concentrated by ultra-/diafiltration (UF/DF) using amembrane made of regenerated cellulose with an appropriate molecularweight cut off (Millipore).

The conjugate prepared by use of this procedure is analyticallycharacterized by measuring total protein, biological activity, anddetermination of the polysialyation degree by measuring the PSA content(resorcinol assay).

Method 3:

Tumor necrosis factor-alpha (TNF-alpha) is transferred into reactionbuffer (e.g. 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride,pH 6.0) and diluted to obtain a protein concentration of 1 mg/ml. A50-fold molar excess of aminooxy-PSA reagent with a MW of 20 kD(described above) is added followed by m-toluidine as a nucleophiliccatalyst (10 mM final concentration) and NaIO₄ (final concentration: 400μM). The coupling reaction is performed for 2 hours in the dark undergentle shaking at room temperature. Subsequently, the reaction isquenched with cysteine for 60 min at RT (cysteine concentration: 10 mM).Then the conductivity of the reaction mixture is adjusted by adding abuffer containing ammonium acetate (50 mM Hepes, 350 mM sodium chloride,5 mM calcium chloride, 8 M ammonium acetate, pH 6.9) and loaded onto acolumn filled with Phenyl Sepharose FF (GE Healthcare, Fairfield, Conn.)pre-equilibrated with 50 mM Hepes, 2.5 M ammonium acetate, 350 mM sodiumchloride, 5 mM calcium chloride, 0.01% Tween 80, pH 6.9. Subsequentlythe conjugate is eluted with 50 mM Hepes, 5 mM calcium chloride, pH 7.5.Finally the PSA-TNF-alpha-containing fractions are collected andsubjected to UF/DF by use of a membrane made of regenerated cellulose(Millipore). The preparation is analytically characterized by measuringtotal protein (Bradford) and biological activity according to methodsknown in the art.

In an alternative embodiment, Method 3 is carried out as follows. Tumornecrosis factor-alpha (TNF-alpha) is transferred into reaction buffer(e.g. 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH6.0) and diluted to obtain a protein concentration of 1 mg/ml. A 50-foldmolar excess of aminooxy-PSA reagent with a MW of 20 kD (describedabove) is added followed by m-toluidine as a nucleophilic catalyst (10mM final concentration) and NaIO₄ (final concentration: 400 μM). Thecoupling reaction is performed for 2 hours in the dark under gentleshaking at room temperature. Subsequently, the reaction is quenched withcysteine for 60 min at RT (cysteine concentration: 10 mM). and theconjugate is purified by ion exchange chromatography. The PSA-TNF-alphacontaining fractions of the eluate are collected and subjected to UF/DFby use of a membrane made of regenerated cellulose (Millipore). Thepreparation is analytically characterized by measuring total protein(Bradford) and biological activity according to methods known in theart.

Method 4:

TNF-alpha is dissolved in or transferred to a reaction buffer (e.g. 50mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to geta final protein concentration of 1.0+/−0.25 mg/ml. Then the pH of thesolution is corrected to 6.0 by drop wise addition of a 0.5 N aqueousHCl solution.

Subsequently the aminooxy-polysialic acid (PSA-ONH₂) reagent is added ina 50-fold molar excess to this TNF-alpha-solution within a maximum timeperiod (t) of 15 minutes under gentle stirring. Then an aqueousm-toluidine solution (50 mM) is added within 15 minutes to get a finalconcentration of 10 mM. Finally a 40 mM aqueous sodium periodatesolution is added to give a concentration of 400 μM.

The reaction mixture is incubated for 120+/−10 min. in the dark at atemperature (T) of T=+22+/−2° C. under gentle shaking. Then the reactionis stopped by the addition of an aqueous L-cysteine solution (1 M) togive a final concentration of 10 mM in the reaction mixture andincubation for 60+/−5 min.

The obtained TNF-alpha conjugate is purified by ion-exchangechromatography. The PSA-TNF-alpha containing fractions of the eluate arecollected and concentrated by ultra-/diafiltration (UF/DF) using amembrane made of regenerated cellulose (Millipore).

The conjugates prepared by use of this procedure are analyticallycharacterized by measuring total protein, biological activity accordingto methods known in the art, and determination of the polysialyationdegree by measuring the PSA content (resorcinol assay).

Example 25 Polysialylation of Insulin Using Aminooxy-PSA and m-Toluidineas a Nucleophilic Catalyst

Method 1:

As described herein, the amino acid sequence of insulin is firstmodified to incorporate at least one glycosylation site. Followingpurification, insulin is glycosylated in vitro according to methodsknown in the art. A starting concentration of insulin is transferredinto a reaction buffer (e.g., 50 mM Hepes, 350 mM sodium chloride, 5 mMcalcium chloride, pH 6.0) and diluted to obtain a protein concentrationof 1 mg/ml. To this solution, NaIO₄ is added to give a finalconcentration of 200 μM. The oxidation is carried at RT for 30 min inthe dark under gentle shaking. The reaction is then quenched withcysteine (final concentration: 10 mM) for 60 min at RT.

The solution is next subjected to UF/DF employing Vivaspin centrifugalfiltrators to remove excess periodate, quencher and the byproductsthereof or, in the alternative, to an IEX column with a volume of 20 ml(Merck EMD TMAE (M)) which is equilibrated with Buffer A (20 mM Hepes, 5mM CaCl₂, pH 7.0). The column is equilibrated with 5 CV Buffer A. Theoxidized insulin is eluted with Buffer B (20 mM Hepes, 5 mM CaCl₂, 1 MNaCl, pH 7.0). The insulin containing fractions are collected. Theprotein content is determined (Coomassie, Bradford) and adjusted to 1mg/ml with reaction buffer and adjusted to pH 6.0 by dropwise additionof 0.5 M HCl.

A 50-fold molar excess of aminooxy-PSA reagent with a MW of 20 kD(described above) is added followed by m-toluidine as a nucleophiliccatalyst (final concentration: 10 mM). The coupling reaction isperformed for 2 hours in the dark under gentle shaking at roomtemperature. The excess of aminooxy reagent is removed by means of HIC.The conductivity of the reaction mixture is adjusted by adding a buffercontaining ammonium acetate (50 mM Hepes, 350 mM sodium chloride, 5 mMcalcium chloride, 8 M ammonium acetate, pH 6.9) and loaded onto a columnfilled with 80 ml Phenyl Sepharose FF (GE Healthcare, Fairfield, Conn.)pre-equilibrated with 50 mM Hepes, 2.5 M ammonium acetate, 350 mM sodiumchloride, 5 mM calcium chloride, pH 6.9. Subsequently the conjugate iseluted with 50 mM Hepes buffer pH 7.5 containing 5 mM CaCl₂. Finally thePSA-insulin containing fractions are collected and subjected to UF/DF byuse of a membrane made of regenerated cellulose (Millipore). Thepreparation is next analytically characterized by measuring totalprotein (Coomassie, Bradford) and biological activity according tomethods known in the art.

In an alternative embodiment, Method 1 is carried out as follows. Asdescribed herein, the amino acid sequence of insulin is first modifiedto incorporate at least one glycosylation site. Following purification,insulin is glycosylated in vitro according to methods known in the art.Insulin is transferred into a reaction buffer (e.g., 50 mM Hepes, 350 mMsodium chloride, 5 mM calcium chloride, pH 6.0) and diluted to obtain aprotein concentration of 1 mg/ml. To this solution, NaIO₄ is added togive a final concentration of 200 μM. The oxidation is carried at RT for30 min in the dark under gentle shaking. The reaction is then quenchedwith cysteine (final concentration: 10 mM) for 60 min at RT.

The solution is next subjected to UF/DF employing Vivaspin centrifugalfiltrators to remove excess periodate, quencher and the byproductsthereof.

A 50-fold molar excess of aminooxy-PSA reagent with a MW of 20 kD(described above) is added followed by m-toluidine as a nucleophiliccatalyst (final concentration: 10 mM). The coupling reaction isperformed for 2 hours in the dark under gentle shaking at roomtemperature. The excess of aminooxy reagent is removed by means of ionexchange chromatography. The PSA-insulin containing fractions of theeluate are collected and subjected to UF/DF by use of a membrane made ofregenerated cellulose (Millipore). The preparation is next analyticallycharacterized by measuring total protein (Coomassie, Bradford) andbiological activity according to methods known in the art.

Method 2:

As described herein, the amino acid sequence of insulin is firstmodified to incorporate at least one glycosylation site. Followingpurification, insulin is glycosylated in vitro according to methodsknown in the art.

Insulin is transferred or dissolved in reaction buffer (e.g. 50 mMHepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to get afinal protein concentration of 1.0+/−0.25 mg/ml. Then the pH of thesolution is corrected to 6.0 by drop wise addition of a 0.5 N aqueousHCl solution. Subsequently, a 40 mM aqueous sodium periodate solution isadded within 10 minutes to give a concentration of 200 μM. The oxidationreaction is carried out for 30+/−5 min at a temperature (T) ofT=+22+/−2° C. Then the reaction is stopped by addition of an aqueousL-cysteine solution (1 M) within 15 minutes at T=+22+/−2° C. to give afinal concentration of 10 mM in the reaction mixture and incubation for60+/−5 min.

The oxidized insulin is further purified by ion exchange chromatography.The oxidized insulin containing fractions of the eluate are collectedand used for the conjugation reaction.

The aminooxy-polysialic acid (PSA-ONH₂) reagent is added in a 50-foldmolar excess to the eluate containing the purified oxidized insulinwithin a maximum time period (t) of 15 minutes under gentle stirring.Then an aqueous m-toluidine solution (50 mM) is added within 15 minutesto get a final concentration of 10 mM. The reaction mixture is incubatedfor 120+/−10 min. at pH 6.0 in the dark at a temperature (T) ofT=+22+/−2° C. under gentle shaking (protein concentration: 1 mg/ml).

The obtained PSA-insulin conjugate is further purified by ion exchangechromatography. The PSA-insulin conjugate containing fractions arecollected and concentrated by ultra-/diafiltration (UF/DF) using amembrane made of regenerated cellulose with an appropriate molecularweight cut off (Millipore).

The conjugate prepared by use of this procedure is analyticallycharacterized by measuring total protein, biological activity, anddetermination of the polysialyation degree by measuring the PSA content(resorcinol assay).

Method 3:

As described herein, the amino acid sequence of insulin is firstmodified to incorporate at least one glycosylation site. Followingpurification, insulin is glycosylated in vitro according to methodsknown in the art.

Insulin is transferred into reaction buffer (50 mM Hepes, 350 mM sodiumchloride, 5 mM calcium chloride, pH 6.0) and diluted to obtain a proteinconcentration of 1 mg/ml. A 50-fold molar excess of aminooxy-PSA reagentwith a MW of 20 kD (described above) is added followed by m-toluidine asa nucleophilic catalyst (10 mM final concentration) and NaIO₄ (finalconcentration: 400 μM). The coupling reaction is performed for 2 hoursin the dark under gentle shaking at room temperature. Subsequently, thereaction is quenched with cysteine for 60 min at RT (cysteineconcentration: 10 mM). Then the conductivity of the reaction mixture isadjusted by adding a buffer containing ammonium acetate (50 mM Hepes,350 mM sodium chloride, 5 mM calcium chloride, 8 M ammonium acetate, pH6.9) and loaded onto a column filled with Phenyl Sepharose FF (GEHealthcare, Fairfield, Conn.) pre-equilibrated with 50 mM Hepes, 2.5 Mammonium acetate, 350 mM sodium chloride, 5 mM calcium chloride, 0.01%Tween 80, pH 6.9. Subsequently the conjugate is eluted with 50 mM Hepes,5 mM calcium chloride, pH 7.5. Finally, the PSA-insulin containingfractions are collected and subjected to UF/DF by use of a membrane madeof regenerated cellulose (Millipore). The preparation is analyticallycharacterized by measuring total protein (Bradford) and biologicalactivity according to methods known in the art.

In an alternative embodiment, Method 3 is carried out as follows. Asdescribed herein, the amino acid sequence of insulin is first modifiedto incorporate at least one glycosylation site. Following purification,insulin is glycosylated in vitro according to methods known in the art.

Insulin is transferred into reaction buffer (e.g. 50 mM Hepes, 350 mMsodium chloride, 5 mM calcium chloride, pH 6.0) and diluted to obtain aprotein concentration of 1 mg/ml. A 50-fold molar excess of aminooxy-PSAreagent with a MW of 20 kD (described above) is added followed bym-toluidine as a nucleophilic catalyst (10 mM final concentration) andNaIO₄ (final concentration: 400 μM). The coupling reaction is performedfor 2 hours in the dark under gentle shaking at room temperature.Subsequently, the reaction is quenched with cysteine for 60 min at RT(cysteine concentration: 10 mM) and the conjugate is purified by ionexchange chromatography. PSA-insulin containing fractions of the eluateare collected and subjected to UF/DF by use of a membrane made ofregenerated cellulose (Millipore). The preparation is analyticallycharacterized by measuring total protein (Bradford) and biologicalactivity according to methods known in the art.

Method 4:

As described herein, the amino acid sequence of insulin is firstmodified to incorporate at least one glycosylation site. Followingpurification, insulin is glycosylated in vitro according to methodsknown in the art.

Insulin is dissolved in or transferred to a reaction buffer (e.g. 50 mMHepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to get afinal protein concentration of 1.0+/−0.25 mg/ml. Then the pH of thesolution is corrected to 6.0 by drop wise addition of a 0.5 N aqueousHCl solution.

Subsequently, the aminooxy-polysialic acid (PSA-ONH2) reagent is addedin a 50-fold molar excess to this insulin-solution within a maximum timeperiod (t) of 15 minutes under gentle stirring. Then an aqueousm-toluidine solution (50 mM) is added within 15 minutes to get a finalconcentration of 10 mM. Finally a 40 mM aqueous sodium periodatesolution is added to give a concentration of 400 μM.

The reaction mixture is incubated for 120+/−10 min. in the dark at atemperature (T) of T=+22+/−2° C. under gentle shaking. Then the reactionis stopped by the addition of an aqueous L-cysteine solution (1 M) togive a final concentration of 10 mM in the reaction mixture andincubation for 60+/−5 min.

The obtained insulin conjugate is purified by ion-exchangechromatography. The PSA-insulin containing fractions of the eluate arecollected and concentrated by ultra-/diafiltration (UF/DF) using amembrane made of regenerated cellulose (Millipore).

The conjugates prepared by use of this procedure are analyticallycharacterized by measuring total protein, biological activity accordingto methods known in the art, and determination of the polysialyationdegree by measuring the PSA content (resorcinol assay).

Example 26 Polysialylation of Interferon-Alpha Using Aminooxy-PSA andm-Toluidine as a Nucleophilic Catalyst

Method 1:

A starting concentration of interferon-alpha is transferred into areaction buffer (e.g., 50 mM Hepes, 350 mM sodium chloride, 5 mM calciumchloride, pH 6.0) and diluted to obtain a protein concentration of 1mg/ml. To this solution, NaIO₄ is added to give a final concentration of200 μM. The oxidation is carried at RT for 30 min in the dark undergentle shaking. The reaction is then quenched with cysteine (finalconcentration: 10 mM) for 60 min at RT.

The solution is next subjected to UF/DF employing Vivaspin centrifugalfiltrators to remove excess periodate, quencher and the byproductsthereof or, in the alternative, to an IEX column with a volume of 20 ml(Merck EMD TMAE (M)) which is equilibrated with Buffer A (20 mM Hepes, 5mM CaCl₂, pH 7.0). The column is equilibrated with 5 CV Buffer A. Theoxidized interferon-alpha is eluted with Buffer B (20 mM Hepes, 5 mMCaCl₂, 1M NaCl, pH 7.0). The interferon-alpha containing fractions arecollected. The protein content is determined (Coomassie, Bradford) andadjusted to 1 mg/ml with reaction buffer and adjusted to pH 6.0 bydropwise addition of 0.5 M HCl.

A 50-fold molar excess of aminooxy-PSA reagent with a MW of 20 kD(described above) is added followed by m-toluidine as a nucleophiliccatalyst (final concentration: 10 mM). The coupling reaction isperformed for 2 hours in the dark under gentle shaking at roomtemperature. The excess of aminooxy reagent is removed by means of HIC.The conductivity of the reaction mixture is adjusted by adding a buffercontaining ammonium acetate (50 mM Hepes, 350 mM sodium chloride, 5 mMcalcium chloride, 8 M ammonium acetate, pH 6.9) and loaded onto a columnfilled with 80 ml Phenyl Sepharose FF (GE Healthcare, Fairfield, Conn.)pre-equilibrated with 50 mM Hepes, 2.5 M ammonium acetate, 350 mM sodiumchloride, 5 mM calcium chloride, pH 6.9. Subsequently, the conjugate iseluted with 50 mM Hepes buffer pH 7.5 containing 5 mM CaCl₂. Finally thePSA-interferon-alpha containing fractions are collected and subjected toUF/DF by use of a membrane made of regenerated cellulose (Millipore).The preparation is next analytically characterized by measuring totalprotein (Coomassie, Bradford) and biological activity according tomethods known in the art.

In an alternative embodiment, Method 1 is carried out as follows.Interferon-alpha is transferred into a reaction buffer (e.g. 50 mMHepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) anddiluted to obtain a protein concentration of 1 mg/ml. To this solution,NaIO₄ is added to give a final concentration of 200 μM. The oxidation iscarried at RT for 30 min in the dark under gentle shaking. The reactionis then quenched with cysteine (final concentration: 10 mM) for 60 minat RT.

The solution is next subjected to UF/DF employing Vivaspin centrifugalfiltrators to remove excess periodate, quencher and the byproductsthereof.

A 50-fold molar excess of aminooxy-PSA reagent with a MW of 20 kD(described above) is added followed by m-toluidine as a nucleophiliccatalyst (final concentration: 10 mM). The coupling reaction isperformed for 2 hours in the dark under gentle shaking at roomtemperature. The excess of aminooxy reagent is removed by means ofion-exchange chromatography. The PSA-interferon-alpha containingfractions of the eluate are collected and subjected to UF/DF by use of amembrane made of regenerated cellulose (Millipore). The preparation isnext analytically characterized by measuring total protein (Coomassie,Bradford) and biological activity according to methods known in the art.

Method 2:

Interferon-alpha is transferred or dissolved in reaction buffer (e.g. 50mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to geta final protein concentration of 1.0+/−0.25 mg/ml. Then the pH of thesolution is corrected to 6.0 by drop wise addition of a 0.5 N aqueousHCl solution. Subsequently, a 40 mM aqueous sodium periodate solution isadded within 10 minutes to give a concentration of 200 μM. The oxidationreaction is carried out for 30+/−5 min at a temperature (T) ofT=+22+/−2° C. Then the reaction is stopped by addition of an aqueousL-cysteine solution (1 M) within 15 minutes at T=+22+/−2° C. to give afinal concentration of 10 mM in the reaction mixture and incubation for60+/−5 min.

The oxidized interferon-alpha is further purified by ion exchangechromatography. The oxidized interferon-alpha containing fractions ofthe eluate are collected and used for the conjugation reaction.

The aminooxy-polysialic acid (PSA-ONH₂) reagent is added in a 50-foldmolar excess to the eluate containing the purified oxidizedinterferon-gamma within a maximum time period (t) of 15 minutes undergentle stirring. Then an aqueous m-toluidine solution (50 mM) is addedwithin 15 minutes to get a final concentration of 10 mM. The reactionmixture is incubated for 120+/−10 min. at pH 6.0 in the dark at atemperature (T) of T=+22+/−2° C. under gentle shaking (proteinconcentration: 1 mg/ml).

The obtained PSA-interferon-alpha conjugate is further purified by ionexchange chromatography. The PSA-interferon-alpha conjugate containingfractions are collected and concentrated by ultra-/diafiltration (UF/DF)using a membrane made of regenerated cellulose with an appropriatemolecular weight cut off (Millipore).

Method 3:

Interferon-alpha is transferred into reaction buffer (50 mM Hepes, 350mM sodium chloride, 5 mM calcium chloride, pH 6.0) and diluted to obtaina protein concentration of 1 mg/ml. A 50-fold molar excess of a PSAaminooxy reagent with a MW of 20 kD (described above) is added followedby m-toluidine as a nucleophilic catalyst (10 mM final concentration)and NaIO₄ (final concentration: 400 μM). The coupling reaction isperformed for 2 hours in the dark under gentle shaking at roomtemperature. Subsequently, the reaction is quenched with cysteine for 60min at RT (cysteine concentration: 10 mM). Then the conductivity of thereaction mixture is adjusted by adding a buffer containing ammoniumacetate (50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, 8 Mammonium acetate, pH 6.9) and loaded onto a column filled with PhenylSepharose FF (GE Healthcare, Fairfield, Conn.) pre-equilibrated with 50mM Hepes, 2.5 M ammonium acetate, 350 mM sodium chloride, 5 mM calciumchloride, 0.01% Tween 80, pH 6.9. Subsequently the conjugate is elutedwith 50 mM Hepes, 5 mM calcium chloride, pH 7.5. Finally, thePSA-interferon-alpha containing fractions are collected and subjected toUF/DF by use of a membrane made of regenerated cellulose (Millipore).The preparation is analytically characterized by measuring total protein(Bradford) and biological activity according to methods known in theart.

In an alternative embodiment, Method 3 is carried out as follows.Interferon-alpha is transferred into reaction buffer (e.g. 50 mM Hepes,350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) and diluted toobtain a protein concentration of 1 mg/ml. A 50-fold molar excess ofaminooxy-PSA reagent with a MW of 20 kD (described above) is addedfollowed by m-toluidine as a nucleophilic catalyst (10 mM finalconcentration) and NaIO₄ (final concentration: 400 μM). The couplingreaction is performed for 2 hours in the dark under gentle shaking atroom temperature. Subsequently, the reaction is quenched with cysteinefor 60 min at RT (cysteine concentration: 10 mM) and the conjugate ispurified by ion exchange chromatography. The PSA-interferon-alphacontaining fractions of the eluate are collected and subjected to UF/DFby use of a membrane made of regenerated cellulose (Millipore). Thepreparation is analytically characterized by measuring total protein(Bradford) and biological activity according to methods known in theart.

Method 4:

Interferon-alpha is dissolved in or transferred to a reaction buffer(e.g. 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH6.0) to get a final protein concentration of 1.0+/−0.25 mg/ml. Then thepH of the solution is corrected to 6.0 by drop wise addition of a 0.5 Naqueous HCl solution.

Subsequently, the aminooxy-polysialic acid (PSA-ONH₂) reagent is addedin a 50-fold molar excess to this interferon-alpha solution within amaximum time period (t) of 15 minutes under gentle stirring. Then anaqueous m-toluidine solution (50 mM) is added within 15 minutes to get afinal concentration of 10 mM. Finally, a 40 mM aqueous sodium periodatesolution is added to give a concentration of 400 μM.

The reaction mixture is incubated for 120+/−10 min. in the dark at atemperature (T) of T=+22+/−2° C. under gentle shaking. Then the reactionis stopped by the addition of an aqueous L-cysteine solution (1 M) togive a final concentration of 10 mM in the reaction mixture andincubation for 60+/−5 min.

The obtained interferon-alpha conjugate is purified by ion-exchangechromatography. The PSA-interferon-alpha containing fractions of theeluate are collected and concentrated by ultra-/diafiltration (UF/DF)using a membrane made of regenerated cellulose (Millipore).

The conjugates prepared by use of this procedure are analyticallycharacterized by measuring total protein, biological activity accordingto methods known in the art, and determination of the polysialyationdegree by measuring the PSA content (resorcinol assay).

Example 27 Polysialylation of Interferon-Gamma Using Aminooxy-PSA andm-Toluidine as a Nucleophilic Catalyst

Method 1:

10 mg interferon-gamma is dissolved in 5 ml histidine buffer, pH 6.0 (20mM L-histidine, 150 mM NaCl). 100 μl of an aqueous sodium periodatesolution (5 mM) is then added and the reaction mixture is incubated for1 h in the dark at 4° C. under gentle stirring and quenched for 15 minat room temperature by the addition of 50 μl of a 1 M aqueous cysteinesolution. The mixture is subsequently subjected to UF/DF employingVivaspin 15R 10 kD centrifugal filtrators to remove excess periodate,quencher and the byproducts thereof.

The retentate (approx. 7 ml), containing oxidized interferon-gamma, ismixed with 2 ml of an aqueous m-toluidine solution (50 mM) and incubatedfor 30 min at room temperature. Then aminooxy-PSA reagent with a MW of20 kD (described above) is added to give a 5-fold molar reagent excess.This mixture is incubated for 2.5 h at RT in the dark under gentlestirring.

The free Interferon-gamma is removed by means of cation exchangechromatography (CEC). The reaction mixture is diluted with 20 ml BufferA (50 mM Hepes, pH 6.5) and loaded onto a 20 ml HiPrep SPFF 16/10 column(GE Healthcare, Fairfield, Conn.) pre-equilibrated with Buffer A. Thenthe column is eluted with Buffer B (50 mM Hepes, 1 M NaCl, pH 6.5). Freeinterferon-gamma is eluted by washing the column with 25% Buffer B andthe conjugate at 50% Buffer B. The conductivity of the conjugatecontaining fractions is subsequently raised to ˜190 mS/cm with Buffer C(50 mM Hepes, 5 M NaCl, pH 6.9) and loaded onto a 20 ml HiPrep Butyl FF16/10 column (GE Healthcare, Fairfield, Conn.) pre-equilibrated withBuffer D (50 mM Hepes, 3 M NaCl, pH 6.9). Free PSA-reagent is washed outwithin 5 CV Buffer D. Subsequently, the conjugate is eluted with 100%Buffer E (50 mM Hepes, pH 6.9). The conjugate containing fractions areconcentrated by UF/DF using a 10 kD membrane made of regeneratedcellulose (88 cm2, cut-off 10 kD, Millipore). The final diafiltrationstep is performed against histidine buffer, pH 6.9 containing 150 mMNaCl. The preparation is analytically characterized by measuring totalprotein (Bradford) and biological activity according to methods known inthe art. For the PSA-Interferon-gamma conjugate a specific activityof >50% in comparison to native Interferon-gamma is determined. Theconjugate is additionally analytically characterized by Size ExclusionHPLC using a Agilent 1200 HPLC system equipped with a Shodex KW 803column under conditions as previously described (Kolarich et al,Transfusion 2006; 46:1959-77). It is shown that the preparation containsno free Interferon gamma.

Method 2:

10 mg interferon-gamma is dissolved in 8 ml histidine buffer, pH 6.0 (20mM L-histidine, 150 mM NaCl). 200 μl of an aqueous sodium periodatesolution (5 mM) and 2 ml of an aqueous m-toluidine solution (50 mM) arethen added. Subsequently the aminooxy-PSA reagent with a MW of 20 kD(described above) is added to give a 5-fold molar reagent excess. Themixture is incubated for 2 h in the dark at room temperature undergentle stirring and quenched for 15 min at room temperature by theaddition of 100 μl of 1 M aqueous cysteine solution.

The free interferon gamma is removed by means of cation exchangechromatography (CEC). The reaction mixture is diluted with 20 ml BufferA (50 mM Hepes, pH 6.5) and loaded onto a 20 ml HiPrep SPFF 16/10 column(GE Healthcare, Fairfield, Conn.) pre-equilibrated with Buffer A. Thenthe column is eluted with Buffer B (50 mM Hepes, 1 M NaCl, pH 6.5). Freeinterferon-gamma is eluted by washing the column with 25% Buffer B andthe conjugate at 50% Buffer B. The conductivity of the conjugatecontaining fractions is subsequently raised to ˜190 mS/cm with Buffer C(50 mM Hepes, 5 M NaCl, pH 6.9) and loaded onto a 20 ml HiPrep Butyl FF16/10 column (GE Healthcare, Fairfield, Conn.) pre-equilibrated withBuffer D (50 mM Hepes, 3 M NaCl, pH 6.9). Free PSA-reagent is washed outwithin 5 CV Buffer D. Subsequently, the conjugate is eluted with 100%Buffer E (50 mM Hepes, pH 6.9). The conjugate containing fractions areconcentrated by UF/DF using a 10 kD membrane made of regeneratedcellulose (88 cm2, cut-off 10 kD/Millipore). The final diafiltrationstep is performed against histidine buffer, pH 6.9 containing 150 mMNaCl. The preparation is analytically characterized by measuring totalprotein (Bradford) and biological activity according to methods known inthe art. For the PSAinterferon-gamma conjugate a specific activityof >50% in comparison to native interferon-gamma is determined. Theconjugate is additionally analytically characterized by Size ExclusionHPLC using a Agilent 1200 HPLC system equipped with a Shodex KW 803column under conditions as previously described (Kolarich et al,Transfusion 2006; 46:1959-77). It is shown that the preparation containsno free interferon-gamma.

Method 3:

10 mg interferon-gamma is dissolved in 8 ml histidine buffer, pH 6.0 (20mM L-histidine, 150 mM NaCl). 200 μl of an aqueous sodium periodatesolution (5 mM) and 2 ml of an aqueous m-toluidine solution (50 mM) arethen added. Subsequently the aminooxy-PSA reagent with a MW of 20 kD(described above) is added to give a 5-fold molar reagent excess. Themixture is incubated for 2 h in the dark at room temperature undergentle stirring and quenched for 15 min at room temperature by theaddition of 100 μl of 1 M aqueous cysteine solution.

The free interferon gamma is removed by means of cation exchangechromatography (CEC). The reaction mixture is diluted with 20 ml BufferA (50 mM Hepes, pH 6.5) and loaded onto a 20 ml HiPrep SPFF 16/10 column(GE Healthcare, Fairfield, Conn.) pre-equilibrated with Buffer A. Thenthe column is eluted with Buffer B (50 mM Hepes, 1 M NaCl, pH 6.5). Freeinterferon-gamma is eluted by washing the column with 25% Buffer B andthe conjugate at 50% Buffer B. The conductivity of the conjugatecontaining fractions is subsequently raised to ˜190 mS/cm with Buffer C(50 mM Hepes, 5 M NaCl, pH 6.9) and loaded onto a 20 ml HiPrep Butyl FF16/10 column (GE Healthcare, Fairfield, Conn.) pre-equilibrated withBuffer D (50 mM Hepes, 3 M NaCl, pH 6.9). Free PSA-reagent is washed outwithin 5 CV Buffer D. Subsequently the conjugate is eluted with 100%Buffer E (50 mM Hepes, pH 6.9). The conjugate containing fractions areconcentrated by UF/DF using a 10 kD membrane made of regeneratedcellulose (88 cm2, cut-off 10 kD/Millipore). The final diafiltrationstep is performed against histidine buffer, pH 6.9 containing 150 mMNaCl. The preparation is analytically characterized by measuring totalprotein (Bradford) and biological activity according to methods known inthe art. For the PSAinterferon-gamma conjugate a specific activityof >50% in comparison to native interferon-gamma is determined. Theconjugate is additionally analytically characterized by Size ExclusionHPLC using a Agilent 1200 HPLC system equipped with a Shodex KW 803column under conditions as previously described (Kolarich et al,Transfusion 2006; 46:1959-77). It is shown that the preparation containsno free interferon-gamma.

Method 4:

Interferon-gamma is dissolved in or transferred to a reaction buffer(e.g. 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH6.0) to get a final protein concentration of 1.0+/−0.25 mg/ml. Then thepH of the solution is corrected to 6.0 by drop wise addition of a 0.5 Naqueous HCl solution.

Subsequently, the aminooxy-polysialic acid (PSA-ONH₂) reagent is addedin a 50-fold molar excess to this interferon-gamma solution within amaximum time period (t) of 15 minutes under gentle stirring. Then anaqueous m-toluidine solution (50 mM) is added within 15 minutes to get afinal concentration of 10 mM. Finally, a 40 mM aqueous sodium periodatesolution is added to give a concentration of 400 μM.

The reaction mixture is incubated for 120+/−10 min. in the dark at atemperature (T) of T=+22+/−2° C. under gentle shaking. Then the reactionis stopped by the addition of an aqueous L-cysteine solution (1 M) togive a final concentration of 10 mM in the reaction mixture andincubation for 60+/−5 min.

The obtained interferon-gamma conjugate is purified by ion-exchangechromatography. The PSA-interferon-gamma containing fractions of theeluate are collected and concentrated by ultra-/diafiltration (UF/DF)using a membrane made of regenerated cellulose (Millipore).

The conjugates prepared by use of this procedure are analyticallycharacterized by measuring total protein, biological activity accordingto methods known in the art, and determination of the polysialyationdegree by measuring the PSA content (resorcinol assay).

Example 28 Polysialylation of G-CSF Using Aminooxy-PSA and m-Toluidineas a Nucleophilic Catalyst

Method 1:

A starting concentration of granulocyte-colony stimulating factor(G-CSF) is transferred into a reaction buffer (e.g., 50 mM Hepes, 350 mMsodium chloride, 5 mM calcium chloride, pH 6.0) and diluted to obtain aprotein concentration of 1 mg/ml. To this solution, NaIO₄ is added togive a final concentration of 200 μM. The oxidation is carried at RT for30 min in the dark under gentle shaking. The reaction is then quenchedwith cysteine (final concentration: 10 mM) for 60 min at RT.

The solution is next subjected to UF/DF employing Vivaspin centrifugalfiltrators to remove excess periodate, quencher and the byproductsthereof or, in the alternative, to an IEX column with a volume of 20 ml(Merck EMD TMAE (M)) which is equilibrated with Buffer A (20 mM Hepes, 5mM CaCl₂, pH 7.0). The column is equilibrated with 5 CV Buffer A. Theoxidized G-CSF is eluted with Buffer B (20 mM Hepes, 5 mM CaCl₂, 1 MNaCl, pH 7.0). The G-CSF containing fractions are collected. The proteincontent is determined (Coomassie, Bradford) and adjusted to 1 mg/ml withreaction buffer and adjusted to pH 6.0 by dropwise addition of 0.5 MHCl.

A 50-fold molar excess of aminooxy-PSA reagent with a MW of 20 kD(described above) is added followed by m-toluidine as a nucleophiliccatalyst (final concentration: 10 mM). The coupling reaction isperformed for 2 hours in the dark under gentle shaking at roomtemperature. The excess of aminooxy reagent is removed by means of HIC.The conductivity of the reaction mixture is adjusted by adding a buffercontaining ammonium acetate (50 mM Hepes, 350 mM sodium chloride, 5 mMcalcium chloride, 8 M ammonium acetate, pH 6.9) and loaded onto a columnfilled with 80 ml Phenyl Sepharose FF (GE Healthcare, Fairfield, Conn.)pre-equilibrated with 50 mM Hepes, 2.5 M ammonium acetate, 350 mM sodiumchloride, 5 mM calcium chloride, pH 6.9. Subsequently, the conjugate iseluted with 50 mM Hepes buffer pH 7.5 containing 5 mM CaCl₂. Finally thePSA-G-CSF-containing fractions are collected and subjected to UF/DF byuse of a membrane made of regenerated cellulose (Millipore). Thepreparation is next analytically characterized by measuring totalprotein (Coomassie, Bradford) and biological activity according tomethods known in the art.

In an alternative embodiment, Method 1 is carried out as follows.Granulocyte-colony stimulating factor (G-CSF) is transferred into areaction buffer (e.g., 50 mM Hepes, 350 mM sodium chloride, 5 mM calciumchloride, pH 6.0) and diluted to obtain a protein concentration of 1mg/ml. To this solution, NaIO₄ is added to give a final concentration of200 μM. The oxidation is carried at RT for 30 min in the dark undergentle shaking. The reaction is then quenched with cysteine (finalconcentration: 10 mM) for 60 min at RT.

The solution is next subjected to UF/DF employing Vivaspin centrifugalfiltrators to remove excess periodate, quencher and the byproductsthereof. A 50-fold molar excess of aminooxy-PSA reagent with a MW of 20kD (described above) is added followed by m-toluidine as a nucleophiliccatalyst (final concentration: 10 mM). The coupling reaction isperformed for 2 hours in the dark under gentle shaking at roomtemperature. The excess of aminooxy reagent is removed by means of ionexchange chromatography. The PSA-G-CSF containing fractions of theeluate are collected and subjected to UF/DF by use of a membrane made ofregenerated cellulose (Millipore). The preparation is next analyticallycharacterized by measuring total protein (Coomassie, Bradford) andbiological activity according to methods known in the art.

Method 2:

G-CSF is transferred or dissolved in reaction buffer (e.g. 50 mM Hepes,350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to get a finalprotein concentration of 1.0+/−0.25 mg/ml. Then the pH of the solutionis corrected to 6.0 by drop wise addition of a 0.5 N aqueous HClsolution. Subsequently, a 40 mM aqueous sodium periodate solution isadded within 10 minutes to give a concentration of 200 μM. The oxidationreaction is carried out for 30+/−5 min at a temperature (T) ofT=+22+/−2° C. Then the reaction is stopped by addition of an aqueousL-cysteine solution (1 M) within 15 minutes at T=+22+/−2° C. to give afinal concentration of 10 mM in the reaction mixture and incubation for60+/−5 min.

The oxidized G-CSF is further purified by ion exchange chromatography.The oxidized G-CSF containing fractions of the eluate are collected andused for the conjugation reaction.

The aminooxy-polysialic acid (PSA-ONH₂) reagent is added in a 50-foldmolar excess to the eluate containing the purified oxidized G-CSF withina maximum time period (t) of 15 minutes under gentle stirring. Then anaqueous m-toluidine solution (50 mM) is added within 15 minutes to get afinal concentration of 10 mM. The reaction mixture is incubated for120+/−10 min. at pH 6.0 in the dark at a temperature (T) of T=+22+/−2°C. under gentle shaking (protein concentration: 1 mg/ml).

The obtained PSA-G-CSF conjugate is further purified by ion exchangechromatography. The PSA-G-CSF conjugate containing fractions arecollected and concentrated by ultra-/diafiltration (UF/DF) using amembrane made of regenerated cellulose with an appropriate molecularweight cut off (Millipore).

The conjugate prepared by use of this procedure is analyticallycharacterized by measuring total protein, biological activity, anddetermination of the polysialyation degree by measuring the PSA content(resorcinol assay).

Method 3:

Granulocyte-colony stimulating factor (G-CSF) is transferred intoreaction buffer (50 mM Hepes, 350 mM sodium chloride, 5 mM calciumchloride, pH 6.0) and diluted to obtain a protein concentration of 1mg/ml. A 50-fold molar excess of aminooxy-PSA reagent with a MW of 20 kD(described above) is added followed by m-toluidine as a nucleophiliccatalyst (10 mM final concentration) and NaIO₄ (final concentration: 400μM). The coupling reaction is performed for 2 hours in the dark undergentle shaking at room temperature. Subsequently, the reaction isquenched with cysteine for 60 min at RT (cysteine concentration: 10 mM).Then the conductivity of the reaction mixture is adjusted by adding abuffer containing ammonium acetate (50 mM Hepes, 350 mM sodium chloride,5 mM calcium chloride, 8 M ammonium acetate, pH 6.9) and loaded onto acolumn filled with Phenyl Sepharose FF (GE Healthcare, Fairfield, Conn.)pre-equilibrated with 50 mM Hepes, 2.5 M ammonium acetate, 350 mM sodiumchloride, 5 mM calcium chloride, 0.01% Tween 80, pH 6.9. Subsequentlythe conjugate is eluted with 50 mM Hepes, 5 mM calcium chloride, pH 7.5.Finally, the PSA-G-CSF-containing fractions are collected and subjectedto UF/DF by use of a membrane made of regenerated cellulose (Millipore).The preparation is analytically characterized by measuring total protein(Bradford) and biological activity according to methods known in theart.

In an alternative embodiment, Method 3 is carried out as follows.Granulocyte-colony stimulating factor (G-CSF) is transferred intoreaction buffer (e.g. 50 mM Hepes, 350 mM sodium chloride, 5 mM calciumchloride, pH 6.0) and diluted to obtain a protein concentration of 1mg/ml. A 50-fold molar excess of aminooxy-PSA reagent with a MW of 20 kD(described above) is added followed by m-toluidine as a nucleophiliccatalyst (10 mM final concentration) and NaIO₄ (final concentration: 400μM). The coupling reaction is performed for 2 hours in the dark undergentle shaking at room temperature. Subsequently, the reaction isquenched with cysteine for 60 min at RT (cysteine concentration: 10 mM)and the conjugate is purified by ion exchange chromatography. ThePSA-G-CSF containing fractions of the eluate are collected and subjectedto UF/DF by use of a membrane made of regenerated cellulose (Millipore).The preparation is analytically characterized by measuring total protein(Bradford) and biological activity according to methods known in theart.

Method 4:

G-CSF is dissolved in or transferred to a reaction buffer (e.g. 50 mMHepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to get afinal protein concentration of 1.0+/−0.25 mg/ml. Then the pH of thesolution is corrected to 6.0 by drop wise addition of a 0.5 N aqueousHCl solution.

Subsequently, the aminooxy-polysialic acid (PSA-ONH₂) reagent is addedin a 50-fold molar excess to this G-CSF solution within a maximum timeperiod (t) of 15 minutes under gentle stirring. Then an aqueousm-toluidine solution (50 mM) is added within 15 minutes to get a finalconcentration of 10 mM. Finally, a 40 mM aqueous sodium periodatesolution is added to give a concentration of 400 μM.

The reaction mixture is incubated for 120+/−10 min. in the dark at atemperature (T) of T=+22+/−2° C. under gentle shaking. Then the reactionis stopped by the addition of an aqueous L-cysteine solution (1 M) togive a final concentration of 10 mM in the reaction mixture andincubation for 60+/−5 min.

The obtained G-CSF conjugate is purified by ion-exchange chromatography.The PSA-G-CSF containing fractions of the eluate are collected andconcentrated by ultra-/diafiltration (UF/DF) using a membrane made ofregenerated cellulose (Millipore).

The conjugates prepared by use of this procedure are analyticallycharacterized by measuring total protein, biological activity accordingto methods known in the art, and determination of the polysialyationdegree by measuring the PSA content (resorcinol assay).

Example 29 Polysialylation of Humira Using Aminooxy-PSA and m-Toluidineas a Nucleophilic Catalyst

Method 1:

A starting concentration of Humira is transferred into a reaction buffer(e.g. 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH6.0) and diluted to obtain a protein concentration of 1 mg/ml. To thissolution, NaIO₄ is added to give a final concentration of 200 μM. Theoxidation is carried at RT for 30 min in the dark under gentle shaking.The reaction is then quenched with cysteine (final concentration: 10 mM)for 60 min at RT.

The solution is next subjected to UF/DF employing Vivaspin centrifugalfiltrators to remove excess periodate, quencher and the byproductsthereof or, in the alternative, to an IEX column with a volume of 20 ml(Merck EMD TMAE (M)) which is equilibrated with Buffer A (20 mM Hepes, 5mM CaCl₂, pH 7.0). The column is equilibrated with 5 CV Buffer A. Theoxidized Humira is eluted with Buffer B (20 mM Hepes, 5 mM CaCl₂, 1MNaCl, pH 7.0). The Humira containing fractions are collected. Theprotein content is determined (Coomassie, Bradford) and adjusted to 1mg/ml with reaction buffer and adjusted to pH 6.0 by dropwise additionof 0.5M HCl.

A 50-fold molar excess of aminooxy-PSA reagent with a MW of 20 kD(described above) is added followed by m-toluidine as a nucleophiliccatalyst (final concentration: 10 mM). The coupling reaction isperformed for 2 hours in the dark under gentle shaking at roomtemperature. The excess of aminooxy reagent is removed by means of HIC.The conductivity of the reaction mixture is adjusted by adding a buffercontaining ammonium acetate (50 mM Hepes, 350 mM sodium chloride, 5 mMcalcium chloride, 8 M ammonium acetate, pH 6.9) and loaded onto a columnfilled with 80 ml Phenyl Sepharose FF (GE Healthcare, Fairfield, Conn.)pre-equilibrated with 50 mM Hepes, 2.5 M ammonium acetate, 350 mM sodiumchloride, 5 mM calcium chloride, pH 6.9. Subsequently the conjugate iseluted with 50 mM Hepes buffer pH 7.5 containing 5 mM CaCl₂. Finally,the PSA-Humira containing fractions are collected and subjected to UF/DFby use of a membrane made of regenerated cellulose (Millipore). Thepreparation is next analytically characterized by measuring totalprotein (Coomassie, Bradford) and biological activity according tomethods known in the art.

In an alternative embodiment, Method 1 is carried out as follows. Humirais transferred into a reaction buffer (e.g. 50 mM Hepes, 350 mM sodiumchloride, 5 mM calcium chloride, pH 6.0) and diluted to obtain a proteinconcentration of 1 mg/ml. To this solution, NaIO₄ is added to give afinal concentration of 200 μM. The oxidation is carried at RT for 30 minin the dark under gentle shaking. The reaction is then quenched withcysteine (final concentration: 10 mM) for 60 min at RT.

The solution is next subjected to UF/DF employing Vivaspin centrifugalfiltrators to remove excess periodate, quencher and the byproductsthereof. A 50-fold molar excess of aminooxy-PSA reagent with a MW of 20kD (described above) is added followed by m-toluidine as a nucleophiliccatalyst (final concentration: 10 mM). The coupling reaction isperformed for 2 hours in the dark under gentle shaking at roomtemperature. The excess of aminooxy reagent is removed by means of ionexchange chromatography The PSA-Humira containing fractions of the eluteare collected and subjected to UF/DF by use of a membrane made ofregenerated cellulose (Millipore). The preparation is next analyticallycharacterized by measuring total protein (Coomassie, Bradford) andbiological activity according to methods known in the art.

Method 2:

Humira is transferred or dissolved in reaction buffer (e.g. 50 mM Hepes,350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to get a finalprotein concentration of 1.0+/−0.25 mg/ml. Then the pH of the solutionis corrected to 6.0 by drop wise addition of a 0.5 N aqueous HClsolution. Subsequently, a 40 mM aqueous sodium periodate solution isadded within 10 minutes to give a concentration of 200 μM. The oxidationreaction is carried out for 30+/−5 min at a temperature (T) ofT=+22+/−2° C. Then the reaction is stopped by addition of an aqueousL-cysteine solution (1 M) within 15 minutes at T=+22+/−2° C. to give afinal concentration of 10 mM in the reaction mixture and incubation for60+/−5 min.

The oxidized Humira is further purified by ion exchange chromatography.The oxidized Humira containing fractions of the eluate are collected andused for the conjugation reaction.

The aminooxy-polysialic acid (PSA-ONH₂) reagent is added in a 50-foldmolar excess to the eluate containing the purified oxidized Humirawithin a maximum time period (t) of 15 minutes under gentle stirring.Then an aqueous m-toluidine solution (50 mM) is added within 15 minutesto get a final concentration of 10 mM. The reaction mixture is incubatedfor 120+/−10 min. at pH 6.0 in the dark at a temperature (T) ofT=+22+/−2° C. under gentle shaking (protein concentration: 1 mg/ml).

The obtained PSA-Humira conjugate is further purified by ion exchangechromatography. The PSA-Humira conjugate containing fractions arecollected and concentrated by ultra-/diafiltration (UF/DF) using amembrane made of regenerated cellulose with an appropriate molecularweight cut off (Millipore).

The conjugate prepared by use of this procedure is analyticallycharacterized by measuring total protein, biological activity, anddetermination of the polysialyation degree by measuring the PSA content(resorcinol assay).

Method 3:

Humira is transferred into reaction buffer (50 mM Hepes, 350 mM sodiumchloride, 5 mM calcium chloride, pH 6.0) and diluted to obtain a proteinconcentration of 1 mg/ml. A 50-fold molar excess of aminooxy-PSA reagentwith a MW of 20 kD (described above) is added followed by m-toluidine asa nucleophilic catalyst (10 mM final concentration) and NaIO₄ (finalconcentration: 400 μM). The coupling reaction is performed for 2 hoursin the dark under gentle shaking at room temperature. Subsequently, thereaction is quenched with cysteine for 60 min at RT (cysteineconcentration: 10 mM). Then the conductivity of the reaction mixture isadjusted by adding a buffer containing ammonium acetate (50 mM Hepes,350 mM sodium chloride, 5 mM calcium chloride, 8 M ammonium acetate, pH6.9) and loaded onto a column filled with Phenyl Sepharose FF (GEHealthcare, Fairfield, Conn.) pre-equilibrated with 50 mM Hepes, 2.5 Mammonium acetate, 350 mM sodium chloride, 5 mM calcium chloride, 0.01%Tween 80, pH 6.9. Subsequently the conjugate is eluted with 50 mM Hepes,5 mM calcium chloride, pH 7.5. Finally the PSA-Humira containingfractions are collected and subjected to UF/DF by use of a membrane madeof regenerated cellulose (Millipore). The preparation is analyticallycharacterized by measuring total protein (Bradford) and biologicalactivity according to methods known in the art.

In an alternative embodiment, Method 3 is carried out as follows. Humirais transferred into reaction buffer (e.g. 50 mM Hepes, 350 mM sodiumchloride, 5 mM calcium chloride, pH 6.0) and diluted to obtain a proteinconcentration of 1 mg/ml. A 50-fold molar excess of aminooxy-PSA reagentwith a MW of 20 kD (described above) is added followed by m-toluidine asa nucleophilic catalyst (10 mM final concentration) and NaIO₄ (finalconcentration: 400 μM). The coupling reaction is performed for 2 hoursin the dark under gentle shaking at room temperature. Subsequently, thereaction is quenched with cysteine for 60 min at RT (cysteineconcentration: 10 mM) and the conjugate is purified by ion exchangechromatography. The PSA-Humira containing fractions of the eluate arecollected and subjected to UF/DF by use of a membrane made ofregenerated cellulose (Millipore). The preparation is analyticallycharacterized by measuring total protein (Bradford) and biologicalactivity according to methods known in the art.

Method 4:

Humira is dissolved in or transferred to a reaction buffer (e.g. 50 mMHepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to get afinal protein concentration of 1.0+/−0.25 mg/ml. Then the pH of thesolution is corrected to 6.0 by drop wise addition of a 0.5 N aqueousHCl solution.

Subsequently, the aminooxy-polysialic acid (PSA-ONH₂) reagent is addedin a 50-fold molar excess to this Humira solution within a maximum timeperiod (t) of 15 minutes under gentle stirring. Then an aqueousm-toluidine solution (50 mM) is added within 15 minutes to get a finalconcentration of 10 mM. Finally a 40 mM aqueous sodium periodatesolution is added to give a concentration of 400 μM.

The reaction mixture is incubated for 120+/−10 min. in the dark at atemperature (T) of T=+22+/−2° C. under gentle shaking. Then the reactionis stopped by the addition of an aqueous L-cysteine solution (1 M) togive a final concentration of 10 mM in the reaction mixture andincubation for 60+/−5 min.

The obtained Humira-conjugate is purified by ion-exchangechromatography. The PSA-Humira containing fractions of the eluate arecollected and concentrated by ultra-/diafiltration (UF/DF) using amembrane made of regenerated cellulose (Millipore).

The conjugates prepared by use of this procedure are analyticallycharacterized by measuring total protein, biological activity accordingto methods known in the art, and determination of the polysialyationdegree by measuring the PSA content (resorcinol assay).

Example 30 Polysialylation of Prolia Using Aminooxy-PSA and m-Toluidineas a Nucleophilic Catalyst

Method 1:

A starting concentration of Prolia is transferred into a reaction buffer(e.g. 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH6.0) and diluted to obtain a protein concentration of 1 mg/ml. To thissolution, NaIO₄ is added to give a final concentration of 200 μM. Theoxidation is carried at RT for 30 min in the dark under gentle shaking.The reaction is then quenched with cysteine (final concentration: 10 mM)for 60 min at RT.

The solution is next subjected to UF/DF employing Vivaspin centrifugalfiltrators to remove excess periodate, quencher and the byproductsthereof or, in the alternative, to an IEX column with a volume of 20 ml(Merck EMD TMAE (M)) which is equilibrated with Buffer A (20 mM Hepes, 5mM CaCl₂, pH 7.0). The column is equilibrated with 5 CV Buffer A. Theoxidized Prolia is eluted with Buffer B (20 mM Hepes, 5 mM CaCl₂, 1MNaCl, pH 7.0). The Prolia containing fractions are collected. Theprotein content is determined (Coomassie, Bradford) and adjusted to 1mg/ml with reaction buffer and adjusted to pH 6.0 by dropwise additionof 0.5 M HCl.

A 50-fold molar excess of aminooxy-PSA reagent with a MW of 20 kD(described above) is added followed by m-toluidine as a nucleophiliccatalyst (final concentration: 10 mM). The coupling reaction isperformed for 2 hours in the dark under gentle shaking at roomtemperature. The excess of aminooxy reagent is removed by means of HIC.The conductivity of the reaction mixture is adjusted by adding a buffercontaining ammonium acetate (50 mM Hepes, 350 mM sodium chloride, 5 mMcalcium chloride, 8 M ammonium acetate, pH 6.9) and loaded onto a columnfilled with 80 ml Phenyl Sepharose FF (GE Healthcare, Fairfield, Conn.)pre-equilibrated with 50 mM Hepes, 2.5 M ammonium acetate, 350 mM sodiumchloride, 5 mM calcium chloride, pH 6.9. Subsequently the conjugate iseluted with 50 mM Hepes buffer pH 7.5 containing 5 mM CaCl₂. Finally,the PSA-Prolia containing fractions are collected and subjected to UF/DFby use of a membrane made of regenerated cellulose (Millipore). Thepreparation is next analytically characterized by measuring totalprotein (Coomassie, Bradford) and biological activity according tomethods known in the art.

In an alternative embodiment, Method 1 is carried out as follows. 10 mgProlia is dissolved in 5 ml histidine buffer, pH 6.0 (20 mM L-histidine,150 mM NaCl). 100 μl of an aqueous sodium periodate solution (5 mM) isthen added and the reaction mixture is incubated for 1 h in the dark at4° C. under gentle stirring and quenched for 15 min at room temperatureby the addition of 50 μl of a 1 M aqueous cysteine solution. The mixtureis subsequently subjected to UF/DF employing Vivaspin 15R 10 kDcentrifugal filtrators to remove excess periodate, quencher and thebyproducts thereof.

The retentate (approx. 7 ml), containing oxidized Prolia, is mixed with2 ml of an aqueous m-toluidine solution (50 mM) and incubated for 30 minat room temperature. Then aminooxy-PSA reagent with a MW of 20 kD(described above) is added to give a 5-fold molar reagent excess. Thismixture is incubated for 2.5 h at RT in the dark under gentle stirring.

The free Prolia is removed by means of cation exchange chromatography(CEC). The reaction mixture is diluted with 20 ml Buffer A (50 mM Hepes,pH 6.5) and loaded onto a 20 ml HiPrep SPFF 16/10 column (GE Healthcare,Fairfield, Conn.) pre-equilibrated with Buffer A. Then the column iseluted with Buffer B (50 mM Hepes, 1 M NaCl, pH 6.5). Free Prolia iseluted by washing the column with 25% Buffer B and the conjugate at 50%Buffer B. The conductivity of the conjugate containing fractions issubsequently raised to ˜190 mS/cm with Buffer C (50 mM Hepes, 5 M NaCl,pH 6.9) and loaded onto a 20 ml HiPrep Butyl FF 16/10 column (GEHealthcare, Fairfield, Conn.) pre-equilibrated with Buffer D (50 mMHepes, 3 M NaCl, pH 6.9). Free PSA-reagent is washed out within 5 CVBuffer D. Subsequently, the conjugate is eluted with 100% Buffer E (50mM Hepes, pH 6.9). The conjugate containing fractions are concentratedby UF/DF using a 10 kD membrane made of regenerated cellulose (88 cm2,cut-off 10 kD, Millipore). The final diafiltration step is performedagainst histidine buffer, pH 6.9 containing 150 mM NaCl. The preparationis analytically characterized by measuring total protein (Bradford) andbiological activity according to methods known in the art. For thePSA-Prolia conjugate a specific activity of >50% in comparison to nativeProlia is determined. The conjugate is additionally analyticallycharacterized by Size Exclusion HPLC using a Agilent 1200 HPLC systemequipped with a Shodex KW 803 column under conditions as previouslydescribed (Kolarich et al, Transfusion 2006; 46:1959-77). It is shownthat the preparation contains no free Prolia.

Method 2:

Prolia is transferred or dissolved in reaction buffer (e.g. 50 mM Hepes,350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to get a finalprotein concentration of 1.0+/−0.25 mg/ml. Then the pH of the solutionis corrected to 6.0 by drop wise addition of a 0.5 N aqueous HClsolution. Subsequently, a 40 mM aqueous sodium periodate solution isadded within 10 minutes to give a concentration of 200 μM. The oxidationreaction is carried out for 30+/−5 min at a temperature (T) ofT=+22+/−2° C. Then the reaction is stopped by addition of an aqueousL-cysteine solution (1 M) within 15 minutes at T=+22+/−2° C. to give afinal concentration of 10 mM in the reaction mixture and incubation for60+/−5 min.

The oxidized Prolia is further purified by ion exchange chromatography.The oxidized Prolia containing fractions of the eluate are collected andused for the conjugation reaction.

The aminooxy-polysialic acid (PSA-ONH₂) reagent is added in a 50-foldmolar excess to the eluate containing the purified oxidized Proliawithin a maximum time period (t) of 15 minutes under gentle stirring.Then an aqueous m-toluidine solution (50 mM) is added within 15 minutesto get a final concentration of 10 mM. The reaction mixture is incubatedfor 120+/−10 min. at pH 6.0 in the dark at a temperature (T) ofT=+22+/−2° C. under gentle shaking (protein concentration: 1 mg/ml).

The obtained Prolia conjugate is further purified by ion exchangechromatography. The Prolia conjugate containing fractions are collectedand concentrated by ultra-/diafiltration (UF/DF) using a membrane madeof regenerated cellulose with an appropriate molecular weight cut off(Millipore).

The conjugate prepared by use of this procedure is analyticallycharacterized by measuring total protein, biological activity, anddetermination of the polysialyation degree by measuring the PSA content(resorcinol assay).

Method 3:

Prolia is transferred into reaction buffer (50 mM Hepes, 350 mM sodiumchloride, 5 mM calcium chloride, pH 6.0) and diluted to obtain a proteinconcentration of 1 mg/ml. A 50-fold molar excess of aminooxy-PSA reagentwith a MW of 20 kD (described above) is added followed by m-toluidine asa nucleophilic catalyst (10 mM final concentration) and NaIO₄ (finalconcentration: 400 μM). The coupling reaction is performed for 2 hoursin the dark under gentle shaking at room temperature. Subsequently, thereaction is quenched with cysteine for 60 min at RT (cysteineconcentration: 10 mM). Then the conductivity of the reaction mixture isadjusted by adding a buffer containing ammonium acetate (50 mM Hepes,350 mM sodium chloride, 5 mM calcium chloride, 8 M ammonium acetate, pH6.9) and loaded onto a column filled with Phenyl Sepharose FF (GEHealthcare, Fairfield, Conn.) pre-equilibrated with 50 mM Hepes, 2.5 Mammonium acetate, 350 mM sodium chloride, 5 mM calcium chloride, 0.01%Tween 80, pH 6.9. Subsequently the conjugate is eluted with 50 mM Hepes,5 mM calcium chloride, pH 7.5. Finally the PSA Prolia-containingfractions are collected and subjected to UF/DF by use of a membrane madeof regenerated cellulose (Millipore). The preparation is analyticallycharacterized by measuring total protein (Bradford) and biologicalactivity according to methods known in the art.

In an alternative embodiment, Method 3 is carried out as follows. 10 mgProlia is dissolved in 8 ml histidine buffer, pH 6.0 (20 mM L-histidine,150 mM NaCl). 200 μl of an aqueous sodium periodate solution (5 mM) and2 ml of an aqueous m-toluidine solution (50 mM) are then added.Subsequently the aminooxy-PSA reagent with a MW of 20 kD (describedabove) is added to give a 5 fold molar reagent excess. The mixture isincubated for 2 h in the dark at room temperature under gentle stirringand quenched for 15 min at room temperature by the addition of 100 μl of1 M aqueous cysteine solution.

The free Prolia is removed by means of cation exchange chromatography(CEC). The reaction mixture is diluted with 20 ml Buffer A (50 mM Hepes,pH 6.5) and loaded onto a 20 ml HiPrep SPFF 16/10 column (GE Healthcare,Fairfield, Conn.) pre-equilibrated with Buffer A. Then the column iseluted with Buffer B (50 mM Hepes, 1 M NaCl, pH 6.5). Free Prolia iseluted by washing the column with 25% Buffer B and the conjugate at 5 0%Buffer B. The conductivity of the conjugate containing fractions issubsequently raised to ˜190 mS/cm with Buffer C (50 mM Hepes, 5 M NaCl,pH 6.9) and loaded onto a 20 ml HiPrep Butyl FF 16/10 column (GEHealthcare, Fairfield, Conn.) pre-equilibrated with Buffer D (50 mMHepes, 3 M NaCl, pH 6.9). Free PSA-reagent is washed out within 5 CVBuffer D. Subsequently the conjugate is eluted with 100% Buffer E (50 mMHepes, pH 6.9). The conjugate containing fractions are concentrated byUF/DF using a 10 kD membrane made of regenerated cellulose (88 cm²,cut-off 10 kD/Millipore). The final diafiltration step is performedagainst histidine buffer, pH 6.9 containing 150 mM NaCl. The preparationis analytically characterized by measuring total protein (Bradford) andbiological activity according to methods known in the art. For thePSA-Prolia conjugate a specific activity of >50% in comparison to nativeProlia is determined. The conjugate is additionally analyticallycharacterized by Size Exclusion HPLC using a Agilent 1200 HPLC systemequipped with a Shodex KW 803 column under conditions as previouslydescribed (Kolarich et al, Transfusion 2006; 46:1959-77). It is shownthat the preparation contains no free Prolia.

Method 4:

Prolia is dissolved in or transferred to a reaction buffer (e.g. 50 mMHepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to get afinal protein concentration of 1.0+/−0.25 mg/ml. Then the pH of thesolution is corrected to 6.0 by drop wise addition of a 0.5 N aqueousHCl solution.

Subsequently the aminooxy-polysialic acid (PSA-ONH₂) reagent is added ina 50-fold molar excess to this Prolia-solution within a maximum timeperiod (t) of 15 minutes under gentle stirring. Then an aqueousm-toluidine solution (50 mM) is added within 15 minutes to get a finalconcentration of 10 mM. Finally a 40 mM aqueous sodium periodatesolution is added to give a concentration of 400 μM.

The reaction mixture is incubated for 120+/−10 min. in the dark at atemperature (T) of T=+22+/−2° C. under gentle shaking. Then the reactionis stopped by the addition of an aqueous L-cysteine solution (1 M) togive a final concentration of 10 mM in the reaction mixture andincubation for 60+/−5 min.

The obtained Prolia conjugate is purified by ion-exchangechromatography. The PSA-Prolia containing fractions of the eluate arecollected and concentrated by ultra-/diafiltration (UF/DF) using amembrane made of regenerated cellulose (Millipore).

The conjugates prepared by use of this procedure are analyticallycharacterized by measuring total protein, biological activity accordingto methods known in the art, and determination of the polysialyationdegree by measuring the PSA content (resorcinol assay).

Example 31 Polysialylation of Other Therapeutic Proteins

Polysialylation reactions performed in the presence of alternativenucleophilic catalysts like m-toluidine or o-aminobenzoic acid asdescribed herein may be extended to other therapeutic proteins. Forexample, in various aspects of the invention, the above polysialylationor PEGylation reactions as described herein with PSA aminooxy or PEGaminooxy reagents is repeated with therapeutic proteins such as thoseproteins described herein.

Example 32 PEGylation of EPO Using an Aminooxy-PEG Reagent andm-Toluidine as a Nucleophilic Catalyst

Method 1:

Erythropoietin (EPO) is PEGylated by use of a linear 20 kD PEGylationreagent containing an aminooxy group. An example of this type of reagentis the Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). EPO isdissolved in 7.0 ml histidine buffer, pH 6.0 (20 mM L-histidine, 150 mMNaCl, 5 mM CaCl₂). An aqueous sodium periodate solution (5 mM) is thenadded and the reaction mixture is incubated for 1 h in the dark at 4° C.under gentle stirring and quenched for 15 min at room temperature by theaddition of 7.5 μl of a 1 M aqueous cysteine solution. The mixture issubsequently subjected to UF/DF employing Vivaspin centrifugalfiltrators to remove excess periodate, quencher and the byproductsthereof.

The retentate containing oxidized EPO is next mixed with an aqueousm-toluidine solution (50 mM) and incubated for 30 min at roomtemperature. aminooxy-PEG reagent with a MW of 20 kD is then added togive a 5-fold molar reagent excess. This mixture is incubated for 2.5 hat room temperature in the dark under gentle stirring.

Finally, the PEG-EPO conjugate is purified by ion-exchangechromatography (e.g. on Q Sepharose FF). For example, 1.5 mg protein/mlgel is loaded on the column equilibrated with 50 mM Hepes buffer, pH 7.4containing 5 mM CaCl₂. The conjugate is eluted with 50 mM Hepes buffercontaining 5 mM CaCl₂ and 500 mM sodium chloride, pH 7.4 and is thensubjected to UF/DF using an appropriate MW cutoff membrane. Thepreparation is next analytically characterized by measuring totalprotein (Coomassie, Bradford) and biological activity according tomethods known in the art.

In an alternative embodiment, Method 1 is carried out as follows. EPO isPEGylated by use of a linear 20 kD PEGylation reagent containing anaminooxy group. An example of this type of reagent is the Sunbright® CAseries from NOF (NOF Corp., Tokyo, Japan). 10 mg EPO is dissolved in 5ml histidine buffer, pH 6.0 (20 mM L-histidine, 150 mM NaCl). 100 μl ofan aqueous sodium periodate solution (5 mM) is then added and thereaction mixture is incubated for 1 h in the dark at 4° C. under gentlestirring and quenched for 15 min at room temperature by the addition of50 μl of a 1 M aqueous cysteine solution. The mixture is subsequentlysubjected to UF/DF employing Vivaspin 15R 10 kD centrifugal filtratorsto remove excess periodate, quencher and the byproducts thereof.

The retentate (approx. 7 ml), containing oxidized EPO, is mixed with 2ml of an aqueous m-toluidine solution (50 mM) and incubated for 30 minat room temperature. Then aminooxy-PEG reagent with a MW of 20 kD(described above) is added to give a 5-fold molar reagent excess. Thismixture is incubated for 2.5 h at RT in the dark under gentle stirring.

Finally, the PEG-EPO conjugate is purified by ion-exchangechromatography on Q Sepharose FF. The reaction mixture is diluted with20 ml Buffer A (50 mM Hepes, pH 7.5) and loaded onto a 20 ml HiPrep QFF16/10 column (GE Healthcare, Fairfield, Conn.) pre-equilibrated withBuffer A. Then the column is eluted with Buffer B (50 mM Hepes, 1 MNaCl, pH 7.5). Free EPO is eluted by washing the column with 25% BufferB and the conjugate at 50% Buffer B. The conjugate containing fractionsare concentrated by UF/DF using a 10 kD membrane made of regeneratedcellulose (88 cm2, cut-off 10 kD/Millipore). The final diafiltrationstep is performed against histidine buffer, pH 7.2 containing 150 mMNaCl. The preparation is analytically characterized by measuring totalprotein (Bradford) and biological activity biological activity accordingto methods known in the art. For the PEG-EPO conjugate a specificactivity of >50% in comparison to native EPO is determined. Theconjugate is additionally analytically characterized by Size ExclusionHPLC using a Agilent 1200 HPLC system equipped with a Shodex KW 803column under conditions as previously described (Kolarich et al,Transfusion 2006; 46:1959-77). It is shown that the preparation containsno free EPO.

Method 2:

EPO is PEGylated by use of a linear 20 kD PEGylation reagent containingan aminooxy group. An example of this type of reagent is the Sunbright®CA series from NOF (NOF Corp., Tokyo, Japan).

EPO is transferred or dissolved in reaction buffer (e.g. 50 mM Hepes,350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to get a finalprotein concentration of 1.0+/−0.25 mg/ml. Then the pH of the solutionis corrected to 6.0 by drop wise addition of a 0.5 N aqueous HClsolution. Subsequently a 40 mM aqueous sodium periodate solution isadded within 10 minutes to give a concentration of 200 μM. The oxidationreaction is carried out for 30+/−5 min at a temperature (T) ofT=+22+/−2° C. Then the reaction is stopped by addition of an aqueousL-cysteine solution (1 M) within 15 minutes at T=+22+/−2° C. to give afinal concentration of 10 mM in the reaction mixture and incubation for60+/−5 min.

The oxidized EPO is further purified by ion exchange chromatography. Theoxidized EPO containing fractions of the eluate are collected and usedfor the conjugation reaction.

The aminooxy-PEG reagent with a MW of 20 kD reagent is added in a50-fold molar excess to the eluate containing the purified oxidized EPOwithin a maximum time period (t) of 15 minutes under gentle stirring.Then an aqueous m-toluidine solution (50 mM) is added within 15 minutesto get a final concentration of 10 mM. The reaction mixture is incubatedfor 120+/−10 min. in the dark at a temperature (T) of T=+22+/−2° C.under gentle shaking.

The obtained PEG-EPO conjugate is further purified by ion exchangechromatography. The PEG-EPO conjugate containing fractions are collectedand concentrated by ultra-/diafiltration (UF/DF) using a membrane madeof regenerated cellulose with an appropriate molecular weight cut off(Millipore).

The conjugate prepared by use of this procedure are analyticallycharacterized by measuring total protein and biological activityaccording to methods known in the art.

Method 3:

EPO is PEGylated by use of a linear 20 kD PEGylation reagent containingan aminooxy group. An example of this type of reagent is the Sunbright®CA series from NOF (NOF Corp., Tokyo, Japan). EPO is dissolved in Hepesbuffer (50 mM Hepes, 150 mM sodium chloride, 5 mM calcium chloride, pH6.0) and mixed with an aqueous sodium periodate solution (10 mM), and anaqueous m-toluidine solution (50 mM). Subsequently, the aminooxy reagentis added to give a 20-fold molar reagent excess. The mixture isincubated for 2 h in the dark at room temperature under gentle stirringand quenched for 15 min at room temperature by the addition of 8 μl ofaqueous cysteine solution (1 M).

Finally, the PEG-EPO conjugate is purified by ion-exchangechromatography on Q Sepharose FF. 1.5 mg protein/ml gel is loaded on thecolumn pre equilibrated with 50 mM Hepes buffer, pH 7.4 containing 5 mMCaCl₂. The conjugate is eluted with 50 mM Hepes buffer containing 5 mMCaCl₂ and 500 mM sodium chloride, pH 7.4 and is then subjected to UF/DFusing a membrane. The preparation is analytically characterized bymeasuring total protein (Bradford) and biological activity according tomethods known in the art.

In an alternative embodiment, Method 3 is carried out as follows. EPO isPEGylated by use of a linear 20 kD PEGylation reagent containing anaminooxy group. An example of this type of reagent is the Sunbright® CAseries from NOF (NOF Corp., Tokyo, Japan). 10 mg EPO is dissolved in ˜8ml histidine buffer, pH 6.0 (20 mM L-histidine, 150 mM NaCl). 200 μl ofan aqueous sodium periodate solution (5 mM) and 2 ml of an aqueousm-toluidine solution (50 mM) are then added. Subsequently, theaminooxy-PEG reagent with a MW of 20 kD (described above) is added togive a 5-fold molar reagent excess. The mixture is incubated for 2 h inthe dark at room temperature under gentle stirring and quenched for 15min at room temperature by the addition of 100 μl of 1 M aqueouscysteine solution.

Finally, the PEG-EPO conjugate is purified by ion-exchangechromatography on Q Sepharose FF. The reaction mixture is diluted with20 ml Buffer A (50 mM Hepes, pH 7.5) and loaded onto a 20 ml HiPrep QFF16/10 column (GE Healthcare, Fairfield, Conn.) pre-equilibrated withBuffer A. Then the column is eluted with Buffer B (50 mM Hepes, 1 MNaCl, pH 7.5). Free EPO is eluted by washing the column with 25% BufferB and the conjugate at 50% Buffer B. The conjugate containing fractionsare concentrated by UF/DF using a 10 kD membrane made of regeneratedcellulose (88 cm2, cut-off 10 kD/Millipore). The final diafiltrationstep is performed against histidine buffer, pH 7.2 containing 150 mMNaCl. The preparation is analytically characterized by measuring totalprotein (Bradford) and biological activity according to methods known inthe art. For the PEG-EPO conjugate a specific activity of >50% incomparison to native EPO is determined. The conjugate is additionallyanalytically characterized by Size Exclusion HPLC using a Agilent 1200HPLC system equipped with a Shodex KW 803 column under conditions aspreviously described (Kolarich et al, Transfusion 2006; 46:1959-77). Itis shown that the preparation contains no free EPO.

Method 4:

EPO is PEGylated by use of a linear 20 kD PEGylation reagent containingan aminooxy group. An example of this type of reagent is the Sunbright®CA series from NOF (NOF Corp., Tokyo, Japan). An initial concentrationor weight of EPO is transferred or dissolved in Hepes buffer (50 mMHepes, 150 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to get afinal protein concentration of 2 mg EPO/ml. Subsequently an 5 mM aqueoussodium periodate solution is added within 15 minutes to give a finalconcentration of 100 μM, followed by addition of an 50 mM aqueousm-toluidine solution to get a final concentration of 10 mM within a timeperiod of 30 minutes. Then the aminooxy-PEG reagent with a MW of 20 kD(described above) is added to give a 20-fold molar reagent excess. Aftercorrection of the pH to 6.0 the mixture is incubated for 2 h in the darkat room temperature under gentle stirring and quenched for 15 min atroom temperature by the addition of a 1 M aqueous L-cysteine solution togive a final concentration of 10 mM.

The PEG-EPO conjugate is purified by means of ion exchangechromatography (IEC). The conjugate containing fractions of the eluateare concentrated by UF/DF using a 10 kD membrane made of regeneratedcellulose (88 cm², cut-off 10 kD/Millipore). The final diafiltrationstep is performed against Hepes buffer (50 mM Hepes, 5 mM CaCl₂, pH7.5).

The preparation is analytically characterized by measuring total protein(Bradford and BCA procedure) and biological activity according to knownmethods.

Example 33 PEGylation of Ang-2 Using an Aminooxy-PEG Reagent andm-Toluidine as a Nucleophilic Catalyst

Method 1:

Ang-2 is PEGylated by use of a linear 20 kD PEGylation reagentcontaining an aminooxy group. An example of this type of reagent is theSunbright® CA series from NOF (NOF Corp., Tokyo, Japan). Ang-2 isdissolved in 7.0 ml histidine buffer, pH 6.0 (20 mM L-histidine, 150 mMNaCl, 5 mM CaCl₂). An aqueous sodium periodate solution (5 mM) is thenadded and the reaction mixture is incubated for 1 h in the dark at 4° C.under gentle stirring and quenched for 15 min at room temperature by theaddition of 7.5 μl of a 1 M aqueous cysteine solution. The mixture issubsequently subjected to UF/DF employing Vivaspin centrifugalfiltrators to remove excess periodate, quencher and the byproductsthereof.

The retentate containing oxidized Ang-2 is next mixed with an aqueousm-toluidine solution (50 mM) and incubated for 30 min at roomtemperature. Aminooxy-PEG reagent with a MW of 20 kD is then added togive a 5-fold molar reagent excess. This mixture is incubated for 2.5 hat room temperature in the dark under gentle stirring.

Finally, the PEG-Ang-2 conjugate is purified by ion-exchangechromatography (e.g. on Q Sepharose FF). For example, 1.5 mg protein/mlgel is loaded on the column equilibrated with 50 mM Hepes buffer, pH 7.4containing 5 mM CaCl₂. The conjugate is eluted with 50 mM Hepes buffercontaining 5 mM CaCl₂ and 500 mM sodium chloride, pH 7.4 and is thensubjected to UF/DF using an appropriate MW cutoff membrane. Thepreparation is next analytically characterized by measuring totalprotein (Coomassie, Bradford) and biological activity according tomethods known in the art.

In an alternative embodiment, Method 1 is carried out as follows. Ang-2is PEGylated by use of a linear 20 kD PEGylation reagent containing anaminooxy group. An example of this type of reagent is the Sunbright® CAseries from NOF (NOF Corp., Tokyo, Japan). Ang-2 is dissolved in 7.0 mlhistidine buffer, pH 6.0 (20 mM L-histidine, 150 mM NaCl, 5 mM CaCl₂).An aqueous sodium periodate solution (5 mM) is then added and thereaction mixture is incubated for 1 h in the dark at 4° C. under gentlestirring and quenched for 15 min at room temperature by the addition of7.5 μl of a 1 M aqueous cysteine solution. The mixture is subsequentlysubjected to UF/DF employing Vivaspin centrifugal filtrators to removeexcess periodate, quencher and the byproducts thereof.

The retentate containing oxidized Ang-2 is next mixed with an aqueousm-toluidine solution (50 mM) and incubated for 30 min at roomtemperature. Aminooxy-PEG reagent with a MW of 20 kD is then added togive a 5-fold molar reagent excess. This mixture is incubated for 2.5 hat room temperature in the dark under gentle stirring.

Finally, the PEG-Ang-2 conjugate is purified by ion-exchangechromatography. The conjugate containing fraction of the eluate arecollected and then subjected to UF/DF using an appropriate MW cutoffmembrane. The preparation is next analytically characterized bymeasuring total protein (Coomassie, Bradford) and biological activityaccording to methods known in the art.

Method 2:

Ang-2 is PEGylated by use of a linear 20 kD PEGylation reagentcontaining an aminooxy group. An example of this type of reagent is theSunbright® CA series from NOF (NOF Corp., Tokyo, Japan).

Ang-2 is transferred or dissolved in reaction buffer (e.g. 50 mM Hepes,350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to get a finalprotein concentration of 1.0+/−0.25 mg/ml. Then the pH of the solutionis corrected to 6.0 by drop wise addition of a 0.5N aqueous HClsolution. Subsequently a 40 mM aqueous sodium periodate solution isadded within 10 minutes to give a concentration of 200 μM. The oxidationreaction is carried out for 30+/−5 min at a temperature (T) ofT=+22+/−2° C. Then the reaction is stopped by addition of an aqueousL-cysteine solution (1 M) within 15 minutes at T=+22+/−2° C. to give afinal concentration of 10 mM in the reaction mixture and incubation for60+/−5 min.

The oxidized Ang-2 is further purified by ion exchange chromatography.The oxidized Ang-2 containing fractions of the eluate are collected andused for the conjugation reaction.

The aminooxy-PEG reagent with a MW of 20 kD reagent is added in a50-fold molar excess to the eluate containing the purified oxidizedAng-2 within a maximum time period (t) of 15 minutes under gentlestirring. Then an aqueous m-toluidine solution (50 mM) is added within15 minutes to get a final concentration of 10 mM. The reaction mixtureis incubated for 120+/−10 min. in the dark at a temperature (T) ofT=+22+/−2° C. under gentle shaking.

The obtained PEG-Ang-2 conjugate is further purified by ion exchangechromatography. The PEG-Ang-2 conjugate containing fractions arecollected and concentrated by ultra-/diafiltration (UF/DF) using amembrane made of regenerated cellulose with an appropriate molecularweight cut off (Millipore).

The conjugate prepared by use of this procedure are analyticallycharacterized by measuring total protein and biological activityaccording to methods known in the art.

Method 3:

Ang-2 is PEGylated by use of a linear 20 kD PEGylation reagentcontaining an aminooxy group. An example of this type of reagent is theSunbright® CA series from NOF (NOF Corp., Tokyo, Japan). Ang-2 isdissolved in Hepes buffer (50 mM Hepes, 150 mM sodium chloride, 5 mMcalcium chloride, pH 6.0) and mixed with an aqueous sodium periodatesolution (10 mM), and an aqueous m-toluidine solution (50 mM).Subsequently the aminooxy reagent is added to give a 20-fold molarreagent excess. The mixture is incubated for 2 h in the dark at roomtemperature under gentle stirring and quenched for 15 min at roomtemperature by the addition of 8 μl of aqueous cysteine solution (1 M).

Finally, the PEG-Ang-2 conjugate is purified by ion-exchangechromatography on Q Sepharose FF. 1.5 mg protein/ml gel is loaded on thecolumn pre equilibrated with 50 mM Hepes buffer, pH 7.4 containing 5 mMCaCl₂. The conjugate is eluted with 50 mM Hepes buffer containing 5 mMCaCl₂ and 500 mM sodium chloride, pH 7.4 and is then subjected to UF/DFusing a membrane. The preparation is analytically characterized bymeasuring total protein (Bradford) and biological activity according tomethods known in the art.

In an alternative embodiment, Method 3 is carried out as follows. Ang-2is PEGylated by use of a linear 20 kD PEGylation reagent containing anaminooxy group. An example of this type of reagent is the Sunbright® CAseries from NOF (NOF Corp., Tokyo, Japan). Ang-2 is dissolved in Hepesbuffer (50 mM Hepes, 150 mM sodium chloride, 5 mM calcium chloride, pH6.0) and mixed with an aqueous sodium periodate solution (10 mM), and anaqueous m-toluidine solution (50 mM). Subsequently the aminooxy reagentis added to give a 20-fold molar reagent excess. The mixture isincubated for 2 h in the dark at room temperature under gentle stirringand quenched for 15 min at room temperature by the addition of 8 μl ofaqueous cysteine solution (1 M).

Finally the PEG-Ang-2 conjugate is purified by ion-exchangechromatography The conjugate containing fractions of the eluate arecollected and then subjected to UF/DF. The preparation is analyticallycharacterized by measuring total protein (Bradford) and biologicalactivity according to methods known in the art.

Method 4:

Ang-2 is PEGylated by use of a linear 20 kD PEGylation reagentcontaining an aminooxy group. An example of this type of reagent is theSunbright® CA series from NOF (NOF Corp., Tokyo, Japan). An initialconcentration or weight of Ang-2 is transferred or dissolved in Hepesbuffer (50 mM Hepes, 150 mM sodium chloride, 5 mM calcium chloride, pH6.0) to get a final protein concentration of 2 mg Ang-2/ml. Subsequentlyan 5 mM aqueous sodium periodate solution is added within 15 minutes togive a final concentration of 100 μM, followed by addition of an 50 mMaqueous m-toluidine solution to get a final concentration of 10 mMwithin a time period of 30 minutes. Then the aminooxy-PEG reagent with aMW of 20 kD (described above) is added to give a 20-fold molar reagentexcess. After correction of the pH to 6.0 the mixture is incubated for 2h in the dark at room temperature under gentle stirring and quenched for15 min at room temperature by the addition of an 1 M aqueous L-cysteinesolution to give a final concentration of 10 mM.

The PEG-Ang-2 conjugate is purified by means of ion exchangechromatography (IEC). The conjugate containing fractions of the eluateare concentrated by UF/DF using a 10 kD membrane made of regeneratedcellulose (88 cm2, cut-off 10 kD/Millipore). The final diafiltrationstep is performed against Hepes buffer (50 mM Hepes, 5 mM CaCl₂, pH7.5).

The preparation is analytically characterized by measuring total protein(Bradford and BCA procedure) and biological activity according to knownmethods.

Subsequently, the free Ang-2 is removed by means of ion exchangechromatography (IEC). The conjugate containing fractions of the eluateare concentrated by UF/DF.

Example 34 PEGylation of VEGF Using an Aminooxy-PEG Reagent andm-Toluidine as a Nucleophilic Catalyst

Method 1:

VEGF is PEGylated by use of a linear 20 kD PEGylation reagent containingan aminooxy group. An example of this type of reagent is the Sunbright®CA series from NOF (NOF Corp., Tokyo, Japan). VEGF is dissolved in 7.0ml histidine buffer, pH 6.0 (20 mM L-histidine, 150 mM NaCl, 5 mMCaCl₂). An aqueous sodium periodate solution (5 mM) is then added andthe reaction mixture is incubated for 1 h in the dark at 4° C. undergentle stirring and quenched for 15 min at room temperature by theaddition of 7.5 μl of a 1 M aqueous cysteine solution. The mixture issubsequently subjected to UF/DF employing Vivaspin centrifugalfiltrators to remove excess periodate, quencher and the byproductsthereof.

The retentate containing oxidized VEGF is next mixed with an aqueousm-toluidine solution (50 mM) and incubated for 30 min at roomtemperature. Aminooxy-PEG reagent with a MW of 20 kD is then added togive a 5-fold molar reagent excess. This mixture is incubated for 2.5 hat room temperature in the dark under gentle stirring.

Finally, the PEG-VEGF conjugate is purified by ion-exchangechromatography (e.g., on Q Sepharose FF). For example, 1.5 mg protein/mlgel is loaded on the column equilibrated with 50 mM Hepes buffer, pH 7.4containing 5 mM CaCl₂. The conjugate is eluted with 50 mM Hepes buffercontaining 5 mM CaCl₂ and 500 mM sodium chloride, pH 7.4 and is thensubjected to UF/DF using an appropriate MW cutoff membrane. Thepreparation is next analytically characterized by measuring totalprotein (Coomassie, Bradford) and biological activity according tomethods known in the art.

In an alternative embodiment, Method 1 is carried out as follows. VEGFis PEGylated by use of a linear 20 kD PEGylation reagent containing anaminooxy group. An example of this type of reagent is the Sunbright® CAseries from NOF (NOF Corp., Tokyo, Japan). VEGF is dissolved in 7.0 mlhistidine buffer, pH 6.0 (20 mM L-histidine, 150 mM NaCl, 5 mM CaCl₂).An aqueous sodium periodate solution (5 mM) is then added and thereaction mixture is incubated for 1 h in the dark at 4° C. under gentlestirring and quenched for 15 min at room temperature by the addition of7.5 μl of a 1 M aqueous cysteine solution. The mixture is subsequentlysubjected to UF/DF employing Vivaspin centrifugal filtrators to removeexcess periodate, quencher and the byproducts thereof.

The retentate containing oxidized VEGF is next mixed with an aqueousm-toluidine solution (50 mM) and incubated for 30 min at roomtemperature. Aminooxy-PEG reagent with a MW of 20 kD is then added togive a 5-fold molar reagent excess. This mixture is incubated for 2.5 hat room temperature in the dark under gentle stirring.

Finally, the PEG-VEGF conjugate is purified by ion-exchangechromatography The conjugate containing fractions of the eluate arecollected and then subjected to UF/DF using an appropriate MW cutoffmembrane. The preparation is next analytically characterized bymeasuring total protein (Coomassie, Bradford) and biological activityaccording to methods known in the art.

Method 2:

VEGF is PEGylated by use of a linear 20 kD PEGylation reagent containingan aminooxy group. An example of this type of reagent is the Sunbright®CA series from NOF (NOF Corp., Tokyo, Japan). VEGF is transferred ordissolved in reaction buffer (e.g. 50 mM Hepes, 350 mM sodium chloride,5 mM calcium chloride, pH 6.0) to get a final protein concentration of1.0+/−0.25 mg/ml. Then the pH of the solution is corrected to 6.0 bydrop wise addition of a 0.5 N aqueous HCl solution. Subsequently, a 40mM aqueous sodium periodate solution is added within 10 minutes to givea concentration of 200 μM. The oxidation reaction is carried out for30+/−5 min at a temperature (T) of T=+22+/−2° C. Then the reaction isstopped by addition of an aqueous L-cysteine solution (1 M) within 15minutes at T=+22+/−2° C. to give a final concentration of 10 mM in thereaction mixture and incubation for 60+/−5 min.

The oxidized VEGF is further purified by ion exchange chromatography.The oxidized VEGF containing fractions of the eluate are collected andused for the conjugation reaction.

The aminooxy-PEG reagent with a MW of 20 kD reagent is added in a50-fold molar excess to the eluate containing the purified oxidized VEGFwithin a maximum time period (t) of 15 minutes under gentle stirring.Then an aqueous m-toluidine solution (50 mM) is added within 15 minutesto get a final concentration of 10 mM. The reaction mixture is incubatedfor 120+/−10 min. in the dark at a temperature (T) of T=+22+/−2° C.under gentle shaking.

The obtained PEG-VEGF conjugate is further purified by ion exchangechromatography. The PEG-VEGF conjugate containing fractions arecollected and concentrated by ultra-/diafiltration (UF/DF) using amembrane made of regenerated cellulose with an appropriate molecularweight cut off (Millipore).

The conjugate prepared by use of this procedure are analyticallycharacterized by measuring total protein and biological activityaccording to methods known in the art.

Method 3:

VEGF is PEGylated by use of a linear 20 kD PEGylation reagent containingan aminooxy group. An example of this type of reagent is the Sunbright®CA series from NOF (NOF Corp., Tokyo, Japan). VEGF is dissolved in Hepesbuffer (50 mM Hepes, 150 mM sodium chloride, 5 mM calcium chloride, pH6.0) and mixed with an aqueous sodium periodate solution (10 mM), and anaqueous m-toluidine solution (50 mM). Subsequently, the aminooxy reagentis added to give a 20-fold molar reagent excess. The mixture isincubated for 2 h in the dark at room temperature under gentle stirringand quenched for 15 min at room temperature by the addition of 8 μl ofaqueous cysteine solution (1 M).

Finally, the PEG-VEGF conjugate is purified by ion-exchangechromatography on Q Sepharose FF. 1.5 mg protein/ml gel is loaded on thecolumn pre equilibrated with 50 mM Hepes buffer, pH 7.4 containing 5 mMCaCl₂. The conjugate is eluted with 50 mM Hepes buffer containing 5 mMCaCl₂ and 500 mM sodium chloride, pH 7.4 and is then subjected to UF/DFusing a membrane. The preparation is analytically characterized bymeasuring total protein (Bradford) and biological activity according tomethods known in the art.

In an alternative embodiment, Method 3 is carried out as follows. VEGFis PEGylated by use of a linear 20 kD PEGylation reagent containing anaminooxy group. An example of this type of reagent is the Sunbright® CAseries from NOF (NOF Corp., Tokyo, Japan). VEGF is dissolved in Hepesbuffer (50 mM Hepes, 150 mM sodium chloride, 5 mM calcium chloride, pH6.0) and mixed with an aqueous sodium periodate solution (10 mM), and anaqueous m-toluidine solution (50 mM). Subsequently, the aminooxy reagentis added to give a 20-fold molar reagent excess. The mixture isincubated for 2 h in the dark at room temperature under gentle stirringand quenched for 15 min at room temperature by the addition of 8 μl ofaqueous cysteine solution (1 M).

Finally, the PEG-VEGF conjugate is purified by ion-exchangechromatography. The conjugate fractions of the eluate are collected andthen subjected to UF/DF. The preparation is analytically characterizedby measuring total protein (Bradford) and biological activity accordingto methods known in the art.

Method 4:

VEGF is PEGylated by use of a linear 20 kD PEGylation reagent containingan aminooxy group. An example of this type of reagent is the Sunbright®CA series from NOF (NOF Corp., Tokyo, Japan). An initial concentrationor weight of VEGF is transferred or dissolved in Hepes buffer (50 mMHepes, 150 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to get afinal protein concentration of 2 mg VEGF/ml. Subsequently, an 5 mMaqueous sodium periodate solution is added within 15 minutes to give afinal concentration of 100 μM, followed by addition of an 50 mM aqueousm-toluidine solution to get a final concentration of 10 mM within a timeperiod of 30 minutes. Then the aminooxy-PEG reagent with a MW of 20 kD(described above) is added to give a 20-fold molar reagent excess. Aftercorrection of the pH to 6.0 the mixture is incubated for 2 h in the darkat room temperature under gentle stirring and quenched for 15 min atroom temperature by the addition of an 1 M aqueous L-cysteine solutionto give a final concentration of 10 mM.

The PEG-VEGF conjugate is purified by means of ion exchangechromatography (IEC). The conjugate containing fractions of the eluateare concentrated by UF/DF using a 10 kD membrane made of regeneratedcellulose (88 cm2, cut-off 10 kD/Millipore). The final diafiltrationstep is performed against Hepes buffer (50 mM Hepes, 5 mM CaCl₂, pH7.5).

The preparation is analytically characterized by measuring total protein(Bradford and BCA procedure) and biological activity according to knownmethods.

Example 35 PEGylation of EGF Using an Aminooxy-PEG Reagent andm-Toluidine as a Nucleophilic Catalyst

Method 1:

EGF is PEGylated by use of a linear 20 kD PEGylation reagent containingan aminooxy group. An example of this type of reagent is the Sunbright®CA series from NOF (NOF Corp., Tokyo, Japan). EGF is dissolved in 7.0 mlhistidine buffer, pH 6.0 (20 mM L-histidine, 150 mM NaCl, 5 mM CaCl₂).An aqueous sodium periodate solution (5 mM) is then added and thereaction mixture is incubated for 1 h in the dark at 4° C. under gentlestirring and quenched for 15 min at room temperature by the addition of7.5 μl of a 1 M aqueous cysteine solution. The mixture is subsequentlysubjected to UF/DF employing Vivaspin centrifugal filtrators to removeexcess periodate, quencher and the byproducts thereof.

The retentate containing oxidized EGF is next mixed with an aqueousm-toluidine solution (50 mM) and incubated for 30 min at roomtemperature. Aminooxy-PEG reagent with a MW of 20 kD is then added togive a 5-fold molar reagent excess. This mixture is incubated for 2.5 hat room temperature in the dark under gentle stirring.

Finally, the PEG-EGF conjugate is purified by ion-exchangechromatography (e.g., on Q Sepharose FF). For example, 1.5 mg protein/mlgel is loaded on the column equilibrated with 50 mM Hepes buffer, pH 7.4containing 5 mM CaCl₂. The conjugate is eluted with 50 mM Hepes buffercontaining 5 mM CaCl₂ and 500 mM sodium chloride, pH 7.4 and is thensubjected to UF/DF using an appropriate MW cutoff membrane. Thepreparation is next analytically characterized by measuring totalprotein (Coomassie, Bradford) and biological activity according tomethods known in the art.

In an alternative embodiment, Method 1 is carried out as follows. EGF isPEGylated by use of a linear 20 kD PEGylation reagent containing anaminooxy group. An example of this type of reagent is the Sunbright® CAseries from NOF (NOF Corp., Tokyo, Japan). EGF is dissolved in 7.0 mlhistidine buffer, pH 6.0 (20 mM L-histidine, 150 mM NaCl, 5 mM CaCl₂).An aqueous sodium periodate solution (5 mM) is then added and thereaction mixture is incubated for 1 h in the dark at 4° C. under gentlestirring and quenched for 15 min at room temperature by the addition of7.5 μl of a 1 M aqueous cysteine solution. The mixture is subsequentlysubjected to UF/DF employing Vivaspin centrifugal filtrators to removeexcess periodate, quencher and the byproducts thereof.

The retentate containing oxidized EGF is next mixed with an aqueousm-toluidine solution (50 mM) and incubated for 30 min at roomtemperature. Aminooxy-PEG reagent with a MW of 20 kD is then added togive a 5-fold molar reagent excess. This mixture is incubated for 2.5 hat room temperature in the dark under gentle stirring.

Finally, the PEG-EGF conjugate is purified by ion-exchangechromatography. The conjugate containing fractions of the eluate arecollected and then subjected to UF/DF using an appropriate MW cutoffmembrane. The preparation is next analytically characterized bymeasuring total protein (Coomassie, Bradford) and biological activityaccording to methods known in the art.

Method 2:

EGF is PEGylated by use of a linear 20 kD PEGylation reagent containingan aminooxy group. An example of this type of reagent is the Sunbright®CA series from NOF (NOF Corp., Tokyo, Japan). EGF is transferred ordissolved in reaction buffer (e.g. 50 mM Hepes, 350 mM sodium chloride,5 mM calcium chloride, pH 6.0) to get a final protein concentration of1.0+/−0.25 mg/ml. Then the pH of the solution is corrected to 6.0 bydrop wise addition of a 0.5 N aqueous HCl solution. Subsequently, a 40mM aqueous sodium periodate solution is added within 10 minutes to givea concentration of 200 μM. The oxidation reaction is carried out for30+/−5 min at a temperature (T) of T=+22+/−2° C. Then the reaction isstopped by addition of an aqueous L-cysteine solution (1 M) within 15minutes at T=+22+/−2° C. to give a final concentration of 10 mM in thereaction mixture and incubation for 60+/−5 min.

The oxidized EGF is further purified by ion exchange chromatography. Theoxidized EGF containing fractions of the eluate are collected and usedfor the conjugation reaction.

The aminooxy-PEG reagent with a MW of 20 kD reagent is added in a50-fold molar excess to the eluate containing the purified oxidized NGFwithin a maximum time period (t) of 15 minutes under gentle stirring.Then an aqueous m-toluidine solution (50 mM) is added within 15 minutesto get a final concentration of 10 mM. The reaction mixture is incubatedfor 120+/−10 min. in the dark at a temperature (T) of T=+22+/−2° C.under gentle shaking.

The obtained PEG-EGF conjugate is further purified by ion exchangechromatography. The PEG-EGF conjugate containing fractions are collectedand concentrated by ultra-/diafiltration (UF/DF) using a membrane madeof regenerated cellulose with an appropriate molecular weight cut off(Millipore).

The conjugate prepared by use of this procedure are analyticallycharacterized by measuring total protein and biological activityaccording to methods known in the art.

Method 3:

EGF is PEGylated by use of a linear 20 kD PEGylation reagent containingan aminooxy group. An example of this type of reagent is the Sunbright®CA series from NOF (NOF Corp., Tokyo, Japan). EGF is dissolved in Hepesbuffer (50 mM Hepes, 150 mM sodium chloride, 5 mM calcium chloride, pH6.0) and mixed with an aqueous sodium periodate solution (10 mM), and anaqueous m-toluidine solution (50 mM). Subsequently the aminooxy reagentis added to give a 20-fold molar reagent excess. The mixture isincubated for 2 h in the dark at room temperature under gentle stirringand quenched for 15 min at room temperature by the addition of 8 μl ofaqueous cysteine solution (1 M).

Finally, the PEG-EGF conjugate is purified by ion-exchangechromatography on Q-Sepharose FF. 1.5 mg protein/ml gel is loaded on thecolumn pre equilibrated with 50 mM Hepes buffer, pH 7.4 containing 5 mMCaCl₂. The conjugate is eluted with 50 mM Hepes buffer containing 5 mMCaCl₂ and 500 mM sodium chloride, pH 7.4 and is then subjected to UF/DFusing a membrane. The preparation is analytically characterized bymeasuring total protein (Bradford) and biological activity according tomethods known in the art.

In an alternative embodiment, Method 3 is carried out as follows. EGF isPEGylated by use of a linear 20 kD PEGylation reagent containing anaminooxy group. An example of this type of reagent is the Sunbright® CAseries from NOF (NOF Corp., Tokyo, Japan). EGF is dissolved in Hepesbuffer (50 mM Hepes, 150 mM sodium chloride, 5 mM calcium chloride, pH6.0) and mixed with an aqueous sodium periodate solution (10 mM), and anaqueous m-toluidine solution (50 mM). Subsequently the aminooxy reagentis added to give a 20-fold molar reagent excess. The mixture isincubated for 2 h in the dark at room temperature under gentle stirringand quenched for 15 min at room temperature by the addition of 8 μl ofaqueous cysteine solution (1 M).

Finally, the PEG-EGF conjugate is purified by ion-exchangechromatography. The conjugate containing fractions of the eluate arecollected and then subjected to UF/DF. The preparation is analyticallycharacterized by measuring total protein (Bradford) and biologicalactivity according to methods known in the art.

Method 4:

EGF is PEGylated by use of a linear 20 kD PEGylation reagent containingan aminooxy group. An example of this type of reagent is the Sunbright®CA series from NOF (NOF Corp., Tokyo, Japan). An initial concentrationor weight of EGF is transferred or dissolved in Hepes buffer (50 mMHepes, 150 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to get afinal protein concentration of 2 mg EGF/ml. Subsequently an 5 mM aqueoussodium periodate solution is added within 15 minutes to give a finalconcentration of 100 μM, followed by addition of an 50 mM aqueousm-toluidine solution to get a final concentration of 10 mM within a timeperiod of 30 minutes. Then the aminooxy-PEG reagent with a MW of 20 kD(described above) is added to give a 20-fold molar reagent excess. Aftercorrection of the pH to 6.0 the mixture is incubated for 2 h in the darkat room temperature under gentle stirring and quenched for 15 min atroom temperature by the addition of an 1 M aqueous L-cysteine solutionto give a final concentration of 10 mM.

The PEG-EGF conjugate is purified by means of ion exchangechromatography (IEC). The conjugate containing fractions of the eluateare concentrated by UF/DF using a 10 kD membrane made of regeneratedcellulose (88 cm2, cut-off 10 kD/Millipore). The final diafiltrationstep is performed against Hepes buffer (50 mM Hepes, 5 mM CaCl₂, pH7.5).

The preparation is analytically characterized by measuring total protein(Bradford and BCA procedure) and biological activity according to knownmethods.

Example 36 PEGylation of NGF Using an Aminooxy-PEG Reagent andm-Toluidine as a Nucleophilic Catalyst

Method 1:

NGF is PEGylated by use of a linear 20 kD PEGylation reagent containingan aminooxy group. An example of this type of reagent is the Sunbright®CA series from NOF (NOF Corp., Tokyo, Japan). NGF is dissolved in 7.0 mlhistidine buffer, pH 6.0 (20 mM L-histidine, 150 mM NaCl, 5 mM CaCl₂).An aqueous sodium periodate solution (5 mM) is then added and thereaction mixture is incubated for 1 h in the dark at 4° C. under gentlestirring and quenched for 15 min at room temperature by the addition of7.5 μl of a 1 M aqueous cysteine solution. The mixture is subsequentlysubjected to UF/DF employing Vivaspin centrifugal filtrators to removeexcess periodate, quencher and the byproducts thereof.

The retentate containing oxidized NGF is next mixed with an aqueousm-toluidine solution (50 mM) and incubated for 30 min at roomtemperature. Aminooxy-PEG reagent with a MW of 20 kD is then added togive a 5-fold molar reagent excess. This mixture is incubated for 2.5 hat room temperature in the dark under gentle stirring.

Finally, the PEG-NGF conjugate is purified by ion-exchangechromatography (e.g., on Q-Sepharose FF). For example, 1.5 mg protein/mlgel is loaded on the column equilibrated with 50 mM Hepes buffer, pH 7.4containing 5 mM CaCl₂. The conjugate is eluted with 50 mM Hepes buffercontaining 5 mM CaCl₂ and 500 mM sodium chloride, pH 7.4 and is thensubjected to UF/DF using an appropriate MW cutoff membrane. Thepreparation is next analytically characterized by measuring totalprotein (Coomassie, Bradford) and biological activity according tomethods known in the art.

In an alternative embodiment, Method 1 is carried out as follows. NGF isPEGylated by use of a linear 20 kD PEGylation reagent containing anaminooxy group. An example of this type of reagent is the Sunbright® CAseries from NOF (NOF Corp., Tokyo, Japan). NGF is dissolved in 7.0 mlhistidine buffer, pH 6.0 (20 mM L-histidine, 150 mM NaCl, 5 mM CaCl₂).An aqueous sodium periodate solution (5 mM) is then added and thereaction mixture is incubated for 1 h in the dark at 4° C. under gentlestirring and quenched for 15 min at room temperature by the addition of7.5 μl of a 1 M aqueous cysteine solution. The mixture is subsequentlysubjected to UF/DF employing Vivaspin centrifugal filtrators to removeexcess periodate, quencher and the byproducts thereof.

The retentate containing oxidized NGF is next mixed with an aqueousm-toluidine solution (50 mM) and incubated for 30 min at roomtemperature. Aminooxy-PEG reagent with a MW of 20 kD is then added togive a 5-fold molar reagent excess. This mixture is incubated for 2.5 hat room temperature in the dark under gentle stirring.

Finally, the PEG-NGF conjugate is purified by ion-exchangechromatography (The conjugate containing fractions of the eluate arecollected and then subjected to UF/DF using an appropriate MW cutoffmembrane. The preparation is next analytically characterized bymeasuring total protein (Coomassie, Bradford) and biological activityaccording to methods known in the art.

Method 2:

NGF is PEGylated by use of a linear 20 kD PEGylation reagent containingan aminooxy group. An example of this type of reagent is the Sunbright®CA series from NOF (NOF Corp., Tokyo, Japan). NGF is transferred ordissolved in reaction buffer (e.g. 50 mM Hepes, 350 mM sodium chloride,5 mM calcium chloride, pH 6.0) to get a final protein concentration of1.0+/−0.25 mg/ml. Then the pH of the solution is corrected to 6.0 bydrop wise addition of a 0.5N aqueous HCl solution. Subsequently a 40 mMaqueous sodium periodate solution is added within 10 minutes to give aconcentration of 200 μM. The oxidation reaction is carried out for30+/−5 min at a temperature (T) of T=+22+/−2° C. Then the reaction isstopped by addition of an aqueous L-cysteine solution (1 M) within 15minutes at T=+22+/−2° C. to give a final concentration of 10 mM in thereaction mixture and incubation for 60+/−5 min.

The oxidized NGF is further purified by ion exchange chromatography. Theoxidized NGF containing fractions of the eluate are collected and usedfor the conjugation reaction.

The aminooxy-PEG reagent with a MW of 20 kD reagent is added in a50-fold molar excess to the eluate containing the purified oxidized NGFwithin a maximum time period (t) of 15 minutes under gentle stirring.Then an aqueous m-toluidine solution (50 mM) is added within 15 minutesto get a final concentration of 10 mM. The reaction mixture is incubatedfor 120+/−10 min. in the dark at a temperature (T) of T=+22+/−2° C.under gentle shaking.

The obtained PEG-NGF conjugate is further purified by ion exchangechromatography. The PEG-NGF conjugate containing fractions are collectedand concentrated by ultra-/diafiltration (UF/DF) using a membrane madeof regenerated cellulose with an appropriate molecular weight cut off(Millipore).

The conjugate prepared by use of this procedure are analyticallycharacterized by measuring total protein and biological activityaccording to methods known in the art.

Method 3:

NGF is PEGylated by use of a linear 20 kD PEGylation reagent containingan aminooxy group. An example of this type of reagent is the Sunbright®CA series from NOF (NOF Corp., Tokyo, Japan). NGF is dissolved in Hepesbuffer (50 mM Hepes, 150 mM sodium chloride, 5 mM calcium chloride, pH6.0) and mixed with an aqueous sodium periodate solution (10 mM), and anaqueous m-toluidine solution (50 mM). Subsequently the aminooxy reagentis added to give a 20-fold molar reagent excess. The mixture isincubated for 2 h in the dark at room temperature under gentle stirringand quenched for 15 min at room temperature by the addition of 8 μl ofaqueous cysteine solution (1 M).

Finally, the PEG-NGF conjugate is purified by ion-exchangechromatography on Q Sepharose FF. 1.5 mg protein/ml gel is loaded on thecolumn pre equilibrated with 50 mM Hepes buffer, pH 7.4 containing 5 mMCaCl₂. The conjugate is eluted with 50 mM Hepes buffer containing 5 mMCaCl₂ and 500 mM sodium chloride, pH 7.4 and is then subjected to UF/DFusing a membrane. The preparation is analytically characterized bymeasuring total protein (Bradford) and biological activity according tomethods known in the art.

In an alternative embodiment, Method 3 is carried out as follows. NGF isPEGylated by use of a linear 20 kD PEGylation reagent containing anaminooxy group. An example of this type of reagent is the Sunbright® CAseries from NOF (NOF Corp., Tokyo, Japan). NGF is dissolved in Hepesbuffer (50 mM Hepes, 150 mM sodium chloride, 5 mM calcium chloride, pH6.0) and mixed with an aqueous sodium periodate solution (10 mM), and anaqueous m-toluidine solution (50 mM). Subsequently the aminooxy reagentis added to give a 20-fold molar reagent excess. The mixture isincubated for 2 h in the dark at room temperature under gentle stirringand quenched for 15 min at room temperature by the addition of 8 μl ofaqueous cysteine solution (1 M).

Finally, the PEG-NGF conjugate is purified by ion-exchangechromatography. The conjugate containing fractions are collected andthen subjected to UF/DF. The preparation is analytically characterizedby measuring total protein (Bradford) and biological activity accordingto methods known in the art.

Method 4:

NGF is PEGylated by use of a linear 20 kD PEGylation reagent containingan aminooxy group. An example of this type of reagent is the Sunbright®CA series from NOF (NOF Corp., Tokyo, Japan). An initial concentrationor weight of NGF is transferred or dissolved in Hepes buffer (50 mMHepes, 150 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to get afinal protein concentration of 2 mg NGF/ml. Subsequently an 5 mM aqueoussodium periodate solution is added within 15 minutes to give a finalconcentration of 100 μM, followed by addition of an 50 mM aqueousm-toluidine solution to get a final concentration of 10 mM within a timeperiod of 30 minutes. Then the aminooxy-PEG reagent with a MW of 20 kD(described above) is added to give a 20-fold molar reagent excess. Aftercorrection of the pH to 6.0 the mixture is incubated for 2 h in the darkat room temperature under gentle stirring and quenched for 15 min atroom temperature by the addition of an 1 M aqueous L-cysteine solutionto give a final concentration of 10 mM.

The PEG-NGF conjugate is purified by means of ion exchangechromatography (IEC). The conjugate containing fractions of the eluateare concentrated by UF/DF using a 10 kD membrane made of regeneratedcellulose (88 cm2, cut-off 10 kD/Millipore). The final diafiltrationstep is performed against Hepes buffer (50 mM Hepes, 5 mM CaCl₂, pH7.5).

The preparation is analytically characterized by measuring total protein(Bradford and BCA procedure) and biological activity according to knownmethods.

Example 37 PEGylation of HGH Using an Aminooxy-PEG Reagent andm-Toluidine as a Nucleophilic Catalyst

Method 1:

As described herein, the amino acid sequence of human growth hormone(HGH) is first modified to incorporate at least one glycosylation site.Following purification, HGH is glycosylated in vitro according tomethods known in the art.

HGH is PEGylated by use of a linear 20 kD PEGylation reagent containingan aminooxy group. An example of this type of reagent is the Sunbright®CA series from NOF (NOF Corp., Tokyo, Japan). HGH is dissolved in 7.0 mlhistidine buffer, pH 6.0 (20 mM L-histidine, 150 mM NaCl, 5 mM CaCl₂).An aqueous sodium periodate solution (5 mM) is then added and thereaction mixture is incubated for 1 h in the dark at 4° C. under gentlestirring and quenched for 15 min at room temperature by the addition of7.5 μl of a 1 M aqueous cysteine solution. The mixture is subsequentlysubjected to UF/DF employing Vivaspin centrifugal filtrators to removeexcess periodate, quencher and the byproducts thereof.

The retentate containing oxidized HGH is next mixed with an aqueousm-toluidine solution (50 mM) and incubated for 30 min at roomtemperature. Aminooxy-PEG reagent with a MW of 20 kD is then added togive a 5-fold molar reagent excess. This mixture is incubated for 2.5 hat room temperature in the dark under gentle stirring.

Finally, the PEG-HGH conjugate is purified by ion-exchangechromatography (e.g., on Q Sepharose FF). For example, 1.5 mg protein/mlgel is loaded on the column equilibrated with 50 mM Hepes buffer, pH 7.4containing 5 mM CaCl₂. The conjugate is eluted with 50 mM Hepes buffercontaining 5 mM CaCl₂ and 500 mM sodium chloride, pH 7.4 and is thensubjected to UF/DF using an appropriate MW cutoff membrane. Thepreparation is next analytically characterized by measuring totalprotein (Coomassie, Bradford) and biological activity according tomethods known in the art.

In an alternative embodiment, Method 1 is carried out as follows. Asdescribed herein, the amino acid sequence of human growth hormone (HGH)is first modified to incorporate at least one glycosylation site.Following purification, HGH is glycosylated in vitro according tomethods known in the art.

HGH is PEGylated by use of a linear 20 kD PEGylation reagent containingan aminooxy group. An example of this type of reagent is the Sunbright®CA series from NOF (NOF Corp., Tokyo, Japan). HGH is dissolved in 7.0 mlhistidine buffer, pH 6.0 (20 mM L-histidine, 150 mM NaCl, 5 mM CaCl₂).An aqueous sodium periodate solution (5 mM) is then added and thereaction mixture is incubated for 1 h in the dark at 4° C. under gentlestirring and quenched for 15 min at room temperature by the addition of7.5 μl of a 1 M aqueous cysteine solution. The mixture is subsequentlysubjected to UF/DF employing Vivaspin centrifugal filtrators to removeexcess periodate, quencher and the byproducts thereof.

The retentate containing oxidized HGH is next mixed with an aqueousm-toluidine solution (50 mM) and incubated for 30 min at roomtemperature. Aminooxy-PEG reagent with a MW of 20 kD is then added togive a 5-fold molar reagent excess. This mixture is incubated for 2.5 hat room temperature in the dark under gentle stirring.

Finally, the PEG-HGH conjugate is purified by ion-exchangechromatography (The conjugate containing fractions of the eluate arecollected and then subjected to UF/DF using an appropriate MW cutoffmembrane. The preparation is next analytically characterized bymeasuring total protein (Coomassie, Bradford) and biological activityaccording to methods known in the art.

Method 2:

As described herein, the amino acid sequence of human growth hormone(HGH) is first modified to incorporate at least one glycosylation site.Following purification, HGH is glycosylated in vitro according tomethods known in the art.

HGH is PEGylated by use of a linear 20 kD PEGylation reagent containingan aminooxy group. An example of this type of reagent is the Sunbright®CA series from NOF (NOF Corp., Tokyo, Japan). HGH is transferred ordissolved in reaction buffer (e.g. 50 mM Hepes, 350 mM sodium chloride,5 mM calcium chloride, pH 6.0) to get a final protein concentration of1.0+/−0.25 mg/ml. Then the pH of the solution is corrected to 6.0 bydrop wise addition of a 0.5N aqueous HCl solution. Subsequently, a 40 mMaqueous sodium periodate solution is added within 10 minutes to give aconcentration of 200 μM. The oxidation reaction is carried out for30+/−5 min at a temperature (T) of T=+22+/−2° C. Then the reaction isstopped by addition of an aqueous L-cysteine solution (1 M) within 15minutes at T=+22+/−2° C. to give a final concentration of 10 mM in thereaction mixture and incubation for 60+/−5 min.

The oxidized HGH is further purified by ion exchange chromatography. Theoxidized HGH containing fractions of the eluate are collected and usedfor the conjugation reaction.

The aminooxy-PEG reagent with a MW of 20 kD reagent is added in a50-fold molar excess to the eluate containing the purified oxidized HGHwithin a maximum time period (t) of 15 minutes under gentle stirring.Then an aqueous m-toluidine solution (50 mM) is added within 15 minutesto get a final concentration of 10 mM. The reaction mixture is incubatedfor 120+/−10 min. in the dark at a temperature (T) of T=+22+/−2° C.under gentle shaking.

The obtained PEG-HGH conjugate is further purified by ion exchangechromatography. The PEG-NGF conjugate containing fractions are collectedand concentrated by ultra-/diafiltration (UF/DF) using a membrane madeof regenerated cellulose with an appropriate molecular weight cut off(Millipore).

The conjugate prepared by use of this procedure are analyticallycharacterized by measuring total protein and biological activityaccording to methods known in the art.

Method 3:

As described herein, the amino acid sequence of human growth hormone(HGH) is first modified to incorporate at least one glycosylation site.Following purification, HGH is glycosylated in vitro according tomethods known in the art.

HGH is PEGylated by use of a linear 20 kD PEGylation reagent containingan aminooxy group. An example of this type of reagent is the Sunbright®CA series from NOF (NOF Corp., Tokyo, Japan). HGH is dissolved in Hepesbuffer (50 mM Hepes, 150 mM sodium chloride, 5 mM calcium chloride, pH6.0) and mixed with an aqueous sodium periodate solution (10 mM), and anaqueous m-toluidine solution (50 mM). Subsequently the aminooxy reagentis added to give a 20-fold molar reagent excess. The mixture isincubated for 2 h in the dark at room temperature under gentle stirringand quenched for 15 min at room temperature by the addition of 8 μl ofaqueous cysteine solution (1 M).

Finally, the PEG-HGH conjugate is purified by ion-exchangechromatography on Q-Sepharose FF. 1.5 mg protein/ml gel is loaded on thecolumn pre equilibrated with 50 mM Hepes buffer, pH 7.4 containing 5 mMCaCl₂. The conjugate is eluted with 50 mM Hepes buffer containing 5 mMCaCl₂ and 500 mM sodium chloride, pH 7.4 and is then subjected to UF/DFusing a membrane. The preparation is analytically characterized bymeasuring total protein (Bradford) and biological activity according tomethods known in the art.

In an alternative embodiment, Method 3 is carried out as follows. Asdescribed herein, the amino acid sequence of human growth hormone (HGH)is first modified to incorporate at least one glycosylation site.Following purification, HGH is glycosylated in vitro according tomethods known in the art. HGH is PEGylated by use of a linear 20 kDPEGylation reagent containing an aminooxy group. An example of this typeof reagent is the Sunbright® CA series from NOF (NOF Corp., Tokyo,Japan). HGH is dissolved in Hepes buffer (50 mM Hepes, 150 mM sodiumchloride, 5 mM calcium chloride, pH 6.0) and mixed with an aqueoussodium periodate solution (10 mM), and an aqueous m-toluidine solution(50 mM). Subsequently the aminooxy reagent is added to give a 20-foldmolar reagent excess. The mixture is incubated for 2 h in the dark atroom temperature under gentle stirring and quenched for 15 min at roomtemperature by the addition of 8 μl of aqueous cysteine solution (1 M).

Finally, the PEG-HGH conjugate is purified by ion-exchangechromatography. The conjugate containing fractions are collected andthen subjected to UF/DF. The preparation is analytically characterizedby measuring total protein (Bradford) and biological activity accordingto methods known in the art.

Method 4:

As described herein, the amino acid sequence of human growth hormone(HGH) is first modified to incorporate at least one glycosylation site.Following purification, HGH is glycosylated in vitro according tomethods known in the art.

HGH is PEGylated by use of a linear 20 kD PEGylation reagent containingan aminooxy group. An example of this type of reagent is the Sunbright®CA series from NOF (NOF Corp., Tokyo, Japan). An initial concentrationor weight of HGH is transferred or dissolved in Hepes buffer (50 mMHepes, 150 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to get afinal protein concentration of 2 mg HGH/ml. Subsequently an 5 mM aqueoussodium periodate solution is added within 15 minutes to give a finalconcentration of 100 μM, followed by addition of an 50 mM aqueousm-toluidine solution to get a final concentration of 10 mM within a timeperiod of 30 minutes. Then the aminooxy-PEG reagent with a MW of 20 kD(described above) is added to give a 20-fold molar reagent excess. Aftercorrection of the pH to 6.0 the mixture is incubated for 2 h in the darkat room temperature under gentle stirring and quenched for 15 min atroom temperature by the addition of a 1 M aqueous L-cysteine solution togive a final concentration of 10 mM.

The PEG-HGH conjugate is purified by means of ion exchangechromatography (IEC). The conjugate containing fractions of the eluateare concentrated by UF/DF using a 10 kD membrane made of regeneratedcellulose (88 cm2, cut-off 10 kD/Millipore). The final diafiltrationstep is performed against Hepes buffer (50 mM Hepes, 5 mM CaCl₂, pH7.5).

The preparation is analytically characterized by measuring total protein(Bradford and BCA procedure) and biological activity according to knownmethods.

Example 38 PEGylation of TNF-Alpha Using an Aminooxy-PEG Reagent andm-Toluidine as a Nucleophilic Catalyst

Method 1:

TNF-alpha is PEGylated by use of a linear 20 kD PEGylation reagentcontaining an aminooxy group. An example of this type of reagent is theSunbright® CA series from NOF (NOF Corp., Tokyo, Japan). TNF-alpha isdissolved in 7.0 ml histidine buffer, pH 6.0 (20 mM L-histidine, 150 mMNaCl, 5 mM CaCl₂). An aqueous sodium periodate solution (5 mM) is thenadded and the reaction mixture is incubated for 1 h in the dark at 4° C.under gentle stirring and quenched for 15 min at room temperature by theaddition of 7.5 μl of a 1 M aqueous cysteine solution. The mixture issubsequently subjected to UF/DF employing Vivaspin centrifugalfiltrators to remove excess periodate, quencher and the byproductsthereof.

The retentate containing oxidized TNF-alpha is next mixed with anaqueous m-toluidine solution (50 mM) and incubated for 30 min at roomtemperature. Aminooxy-PEG reagent with a MW of 20 kD is then added togive a 5-fold molar reagent excess. This mixture is incubated for 2.5 hat room temperature in the dark under gentle stirring.

Finally, the PEG-TNF-alpha conjugate is purified by ion-exchangechromatography (e.g., on Q-Sepharose FF). For example, 1.5 mg protein/mlgel is loaded on the column equilibrated with 50 mM Hepes buffer, pH 7.4containing 5 mM CaCl₂. The conjugate is eluted with 50 mM Hepes buffercontaining 5 mM CaCl₂ and 500 mM sodium chloride, pH 7.4 and is thensubjected to UF/DF using an appropriate MW cutoff membrane. Thepreparation is next analytically characterized by measuring totalprotein (Coomassie, Bradford) and biological activity according tomethods known in the art.

In an alternative embodiment, Method 1 is carried out as follows.TNF-alpha is PEGylated by use of a linear 20 kD PEGylation reagentcontaining an aminooxy group. An example of this type of reagent is theSunbright® CA series from NOF (NOF Corp., Tokyo, Japan). TNF-alpha isdissolved in 7.0 ml histidine buffer, pH 6.0 (20 mM L-histidine, 150 mMNaCl, 5 mM CaCl₂). An aqueous sodium periodate solution (5 mM) is thenadded and the reaction mixture is incubated for 1 h in the dark at 4° C.under gentle stirring and quenched for 15 min at room temperature by theaddition of 7.5 μl of a 1 M aqueous cysteine solution. The mixture issubsequently subjected to UF/DF employing Vivaspin centrifugalfiltrators to remove excess periodate, quencher and the byproductsthereof.

The retentate containing oxidized TNF-alpha is next mixed with anaqueous m-toluidine solution (50 mM) and incubated for 30 min at roomtemperature. Aminooxy-PEG reagent with a MW of 20 kD is then added togive a 5-fold molar reagent excess. This mixture is incubated for 2.5 hat room temperature in the dark under gentle stirring.

Finally, the PEG-TNF-alpha conjugate is purified by ion-exchangechromatography. The conjugate containing fractions of the eluate arecollected and then subjected to UF/DF using an appropriate MW cutoffmembrane. The preparation is next analytically characterized bymeasuring total protein (Coomassie, Bradford) and biological activityaccording to methods known in the art.

Method 2:

TNF-alpha is PEGylated by use of a linear 20 kD PEGylation reagentcontaining an aminooxy group. An example of this type of reagent is theSunbright® CA series from NOF (NOF Corp., Tokyo, Japan). TNF-alpha istransferred or dissolved in reaction buffer (e.g. 50 mM Hepes, 350 mMsodium chloride, 5 mM calcium chloride, pH 6.0) to get a final proteinconcentration of 1.0+/−0.25 mg/ml. Then the pH of the solution iscorrected to 6.0 by drop wise addition of a 0.5N aqueous HCl solution.Subsequently a 40 mM aqueous sodium periodate solution is added within10 minutes to give a concentration of 200 μM. The oxidation reaction iscarried out for 30+/−5 min at a temperature (T) of T=+22+/−2° C. Thenthe reaction is stopped by addition of an aqueous L-cysteine solution (1M) within 15 minutes at T=+22+/−2° C. to give a final concentration of10 mM in the reaction mixture and incubation for 60+/−5 min.

The oxidized TNF-alpha is further purified by ion exchangechromatography. The oxidized TNF-alpha containing fractions of theeluate are collected and used for the conjugation reaction.

The aminooxy-PEG reagent with a MW of 20 kD reagent is added in a50-fold molar excess to the eluate containing the purified oxidized TNFalpha within a maximum time period (t) of 15 minutes under gentlestirring. Then an aqueous m-toluidine solution (50 mM) is added within15 minutes to get a final concentration of 10 mM. The reaction mixtureis incubated for 120+/−10 min. in the dark at a temperature (T) ofT=+22+/−2° C. under gentle shaking.

The obtained PEG-TNF-alpha conjugate is further purified by ion exchangechromatography. The PEG-TNF-alpha conjugate containing fractions arecollected and concentrated by ultra-/diafiltration (UF/DF) using amembrane made of regenerated cellulose with an appropriate molecularweight cut off (Millipore).

The conjugate prepared by use of this procedure are analyticallycharacterized by measuring total protein and biological activityaccording to methods known in the art.

Method 3:

TNF-alpha is PEGylated by use of a linear 20 kD PEGylation reagentcontaining an aminooxy group. An example of this type of reagent is theSunbright® CA series from NOF (NOF Corp., Tokyo, Japan). TNF-alpha isdissolved in Hepes buffer (50 mM Hepes, 150 mM sodium chloride, 5 mMcalcium chloride, pH 6.0) and mixed with an aqueous sodium periodatesolution (10 mM), and an aqueous m-toluidine solution (50 mM).Subsequently the aminooxy reagent is added to give a 20-fold molarreagent excess. The mixture is incubated for 2 h in the dark at roomtemperature under gentle stirring and quenched for 15 min at roomtemperature by the addition of 8 μl of aqueous cysteine solution (1 M).

Finally, the PEG-TNF-alpha conjugate is purified by ion-exchangechromatography on Q-Sepharose FF. 1.5 mg protein/ml gel is loaded on thecolumn pre equilibrated with 50 mM Hepes buffer, pH 7.4 containing 5 mMCaCl₂. The conjugate is eluted with 50 mM Hepes buffer containing 5 mMCaCl₂ and 500 mM sodium chloride, pH 7.4 and is then subjected to UF/DFusing a membrane. The preparation is analytically characterized bymeasuring total protein (Bradford) and biological activity according tomethods known in the art.

In an alternative embodiment, Method 3 is carried out as follows.TNF-alpha is PEGylated by use of a linear 20 kD PEGylation reagentcontaining an aminooxy group. An example of this type of reagent is theSunbright® CA series from NOF (NOF Corp., Tokyo, Japan). TNF-alpha isdissolved in Hepes buffer (50 mM Hepes, 150 mM sodium chloride, 5 mMcalcium chloride, pH 6.0) and mixed with an aqueous sodium periodatesolution (10 mM), and an aqueous m-toluidine solution (50 mM).Subsequently, the aminooxy reagent is added to give a 20-fold molarreagent excess. The mixture is incubated for 2 h in the dark at roomtemperature under gentle stirring and quenched for 15 min at roomtemperature by the addition of 8 μl of aqueous cysteine solution (1 M).

Finally, the PEG-TNF-alpha conjugate is purified by ion-exchangechromatography. The conjugate containing fractions are collected andthen subjected to UF/DF. The preparation is analytically characterizedby measuring total protein (Bradford) and biological activity accordingto methods known in the art.

Method 4:

TNF-alpha is PEGylated by use of a linear 20 kD PEGylation reagentcontaining an aminooxy group. An example of this type of reagent is theSunbright® CA series from NOF (NOF Corp., Tokyo, Japan). An initialconcentration or weight of TNF-alpha is transferred or dissolved inHepes buffer (50 mM Hepes, 150 mM sodium chloride, 5 mM calciumchloride, pH 6.0) to get a final protein concentration of 2 mgTNF-alpha/ml. Subsequently, an 5 mM aqueous sodium periodate solution isadded within 15 minutes to give a final concentration of 100 μM,followed by addition of an 50 mM aqueous m-toluidine solution to get afinal concentration of 10 mM within a time period of 30 minutes. Thenthe aminooxy-PEG reagent with a MW of 20 kD (described above) is addedto give a 20-fold molar reagent excess. After correction of the pH to6.0 the mixture is incubated for 2 h in the dark at room temperatureunder gentle stirring and quenched for 15 min at room temperature by theaddition of an 1 M aqueous L-cysteine solution to give a finalconcentration of 10 mM.

The PEG-TNF-alpha conjugate is purified by means of ion exchangechromatography (IEC). The conjugate containing fractions of the eluateare concentrated by UF/DF using a 10 kD membrane made of regeneratedcellulose (88 cm2, cut-off 10 kD/Millipore). The final diafiltrationstep is performed against Hepes buffer (50 mM Hepes, 5 mM CaCl₂, pH7.5).

The preparation is analytically characterized by measuring total protein(Bradford and BCA procedure) and biological activity according to knownmethods.

Example 39 PEGylation of Insulin Using an Aminooxy-PEG Reagent andm-Toluidine as a Nucleophilic Catalyst

Method 1:

As described herein, the amino acid sequence of insulin is firstmodified to incorporate at least one glycosylation site. Followingpurification, insulin is glycosylated in vitro according to methodsknown in the art. Insulin is PEGylated by use of a linear 20 kDPEGylation reagent containing an aminooxy group. An example of this typeof reagent is the Sunbright® CA series from NOF (NOF Corp., Tokyo,Japan). Insulin is dissolved in 7.0 ml histidine buffer, pH 6.0 (20 mML-histidine, 150 mM NaCl, 5 mM CaCl₂). An aqueous sodium periodatesolution (5 mM) is then added and the reaction mixture is incubated for1 h in the dark at 4° C. under gentle stirring and quenched for 15 minat room temperature by the addition of 7.5 μl of a 1 M aqueous cysteinesolution. The mixture is subsequently subjected to UF/DF employingVivaspin centrifugal filtrators to remove excess periodate, quencher andthe byproducts thereof.

The retentate containing oxidized insulin is next mixed with an aqueousm-toluidine solution (50 mM) and incubated for 30 min at roomtemperature. Aminooxy-PEG reagent with a MW of 20 kD is then added togive a 5-fold molar reagent excess. This mixture is incubated for 2.5 hat room temperature in the dark under gentle stirring.

Finally, the PEG-insulin conjugate is purified by ion-exchangechromatography (e.g., on Q-Sepharose FF). For example, 1.5 mg protein/mlgel is loaded on the column equilibrated with 50 mM Hepes buffer, pH 7.4containing 5 mM CaCl₂. The conjugate is eluted with 50 mM Hepes buffercontaining 5 mM CaCl₂ and 500 mM sodium chloride, pH 7.4 and is thensubjected to UF/DF using an appropriate MW cutoff membrane. Thepreparation is next analytically characterized by measuring totalprotein (Coomassie, Bradford) and biological activity according tomethods known in the art.

In an alternative embodiment, Method 1 is carried out as follows. Asdescribed herein, the amino acid sequence of insulin is first modifiedto incorporate at least one glycosylation site. Following purification,insulin is glycosylated in vitro according to methods known in the art.Insulin is PEGylated by use of a linear 20 kD PEGylation reagentcontaining an aminooxy group. An example of this type of reagent is theSunbright® CA series from NOF (NOF Corp., Tokyo, Japan). Insulin isdissolved in 7.0 ml histidine buffer, pH 6.0 (20 mM L-histidine, 150 mMNaCl, 5 mM CaCl₂). An aqueous sodium periodate solution (5 mM) is thenadded and the reaction mixture is incubated for 1 h in the dark at 4° C.under gentle stirring and quenched for 15 min at room temperature by theaddition of 7.5 μl of a 1 M aqueous cysteine solution. The mixture issubsequently subjected to UF/DF employing Vivaspin centrifugalfiltrators to remove excess periodate, quencher and the byproductsthereof.

The retentate containing oxidized insulin is next mixed with an aqueousm-toluidine solution (50 mM) and incubated for 30 min at roomtemperature. Aminooxy-PEG reagent with a MW of 20 kD is then added togive a 5-fold molar reagent excess. This mixture is incubated for 2.5 hat room temperature in the dark under gentle stirring.

Finally, the PEG-insulin conjugate is purified by ion-exchangechromatography. The conjugate containing fractions of the eluate arecollected and then subjected to UF/DF using an appropriate MW cutoffmembrane. The preparation is next analytically characterized bymeasuring total protein (Coomassie, Bradford) and biological activityaccording to methods known in the art.

Method 2:

As described herein, the amino acid sequence of insulin is firstmodified to incorporate at least one glycosylation site. Followingpurification, insulin is glycosylated in vitro according to methodsknown in the art.

Insulin is PEGylated by use of a linear 20 kD PEGylation reagentcontaining an aminooxy group. An example of this type of reagent is theSunbright® CA series from NOF (NOF Corp., Tokyo, Japan). Insulin istransferred or dissolved in reaction buffer (e.g. 50 mM Hepes, 350 mMsodium chloride, 5 mM calcium chloride, pH 6.0) to get a final proteinconcentration of 1.0+/−0.25 mg/ml. Then the pH of the solution iscorrected to 6.0 by drop wise addition of a 0.5 N aqueous HCl solution.Subsequently, a 40 mM aqueous sodium periodate solution is added within10 minutes to give a concentration of 200 μM. The oxidation reaction iscarried out for 30+/−5 min at a temperature (T) of T=+22+/−2° C. Thenthe reaction is stopped by addition of an aqueous L-cysteine solution (1M) within 15 minutes at T=+22+/−2° C. to give a final concentration of10 mM in the reaction mixture and incubation for 60+/−5 min.

The oxidized insulin is further purified by ion exchange chromatography.The oxidized insulin containing fractions of the eluate are collectedand used for the conjugation reaction.

The aminooxy-PEG reagent with a MW of 20 kD reagent is added in a50-fold molar excess to the eluate containing the purified oxidizedinsulin within a maximum time period (t) of 15 minutes under gentlestirring. Then an aqueous m-toluidine solution (50 mM) is added within15 minutes to get a final concentration of 10 mM. The reaction mixtureis incubated for 120+/−10 min. in the dark at a temperature (T) ofT=+22+/−2° C. under gentle shaking.

The obtained PEG-insulin conjugate is further purified by ion exchangechromatography. The PEG-insulin conjugate containing fractions arecollected and concentrated by ultra-/diafiltration (UF/DF) using amembrane made of regenerated cellulose with an appropriate molecularweight cut off (Millipore).

The conjugate prepared by use of this procedure are analyticallycharacterized by measuring total protein and biological activityaccording to methods known in the art.

Method 3:

As described herein, the amino acid sequence of insulin is firstmodified to incorporate at least one glycosylation site. Followingpurification, insulin is glycosylated in vitro according to methodsknown in the art.

Insulin is PEGylated by use of a linear 20 kD PEGylation reagentcontaining an aminooxy group. An example of this type of reagent is theSunbright® CA series from NOF (NOF Corp., Tokyo, Japan). Insulin isdissolved in Hepes buffer (50 mM Hepes, 150 mM sodium chloride, 5 mMcalcium chloride, pH 6.0) and mixed with an aqueous sodium periodatesolution (10 mM), and an aqueous m-toluidine solution (50 mM).Subsequently, the aminooxy reagent is added to give a 20-fold molarreagent excess. The mixture is incubated for 2 h in the dark at roomtemperature under gentle stirring and quenched for 15 min at roomtemperature by the addition of 8 μl of aqueous cysteine solution (1 M).

Finally, the PEG-insulin conjugate is purified by ion-exchangechromatography on Q Sepharose FF. 1.5 mg protein/ml gel is loaded on thecolumn pre equilibrated with 50 mM Hepes buffer, pH 7.4 containing 5 mMCaCl₂. The conjugate is eluted with 50 mM Hepes buffer containing 5 mMCaCl₂ and 500 mM sodium chloride, pH 7.4 and is then subjected to UF/DFusing a membrane. The preparation is analytically characterized bymeasuring total protein (Bradford) and biological activity according tomethods known in the art.

In an alternative embodiment, Method 3 is carried out as follows. Asdescribed herein, the amino acid sequence of insulin is first modifiedto incorporate at least one glycosylation site. Following purification,insulin is glycosylated in vitro according to methods known in the art.Insulin is PEGylated by use of a linear 20 kD PEGylation reagentcontaining an aminooxy group. An example of this type of reagent is theSunbright® CA series from NOF (NOF Corp., Tokyo, Japan). Insulin isdissolved in Hepes buffer (50 mM Hepes, 150 mM sodium chloride, 5 mMcalcium chloride, pH 6.0) and mixed with an aqueous sodium periodatesolution (10 mM), and an aqueous m-toluidine solution (50 mM).Subsequently the aminooxy reagent is added to give a 20-fold molarreagent excess. The mixture is incubated for 2 h in the dark at roomtemperature under gentle stirring and quenched for 15 min at roomtemperature by the addition of 8 μl of aqueous cysteine solution (1 M).

Finally, the insulin-conjugate is purified by ion-exchangechromatography. The conjugate containing fractions are collected andthen subjected to UF/DF. The preparation is analytically characterizedby measuring total protein (Bradford) and biological activity accordingto methods known in the art.

Method 4:

As described herein, the amino acid sequence of insulin is firstmodified to incorporate at least one glycosylation site. Followingpurification, insulin is glycosylated in vitro according to methodsknown in the art.

Insulin is PEGylated by use of a linear 20 kD PEGylation reagentcontaining an aminooxy group. An example of this type of reagent is theSunbright® CA series from NOF (NOF Corp., Tokyo, Japan). An initialconcentration or weight of insulin is transferred or dissolved in Hepesbuffer (50 mM Hepes, 150 mM sodium chloride, 5 mM calcium chloride, pH6.0) to get a final protein concentration of 2 mg insulin/ml.Subsequently an 5 mM aqueous sodium periodate solution is added within15 minutes to give a final concentration of 100 μM, followed by additionof an 50 mM aqueous m-toluidine solution to get a final concentration of10 mM within a time period of 30 minutes. Then the aminooxy-PEG reagentwith a MW of 20 kD (described above) is added to give a 20-fold molarreagent excess. After correction of the pH to 6.0 the mixture isincubated for 2 h in the dark at room temperature under gentle stirringand quenched for 15 min at room temperature by the addition of a 1 Maqueous L-cysteine solution to give a final concentration of 10 mM.

The PEG-insulin conjugate is purified by means of ion exchangechromatography (IEC). The conjugate containing fractions of the eluateare concentrated by UF/DF using a 10 kD membrane made of regeneratedcellulose (88 cm2, cut-off 10 kD/Millipore). The final diafiltrationstep is performed against Hepes buffer (50 mM Hepes, 5 mM CaCl₂, pH7.5).

The preparation is analytically characterized by measuring total protein(Bradford and BCA procedure) and biological activity according to knownmethods.

Example 40 PEGylation of Interferon-Alpha Using an Aminooxy-PEG Reagentand m-Toluidine as a Nucleophilic Catalyst

Method 1:

Interferon-alpha is PEGylated by use of a linear 20 kD PEGylationreagent containing an aminooxy group. An example of this type of reagentis the Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan).Interferon-alpha is dissolved in 7.0 ml histidine buffer, pH 6.0 (20 mML-histidine, 150 mM NaCl, 5 mM CaCl₂). An aqueous sodium periodatesolution (5 mM) is then added and the reaction mixture is incubated for1 h in the dark at 4° C. under gentle stirring and quenched for 15 minat room temperature by the addition of 7.5 μl of a 1 M aqueous cysteinesolution. The mixture is subsequently subjected to UF/DF employingVivaspin centrifugal filtrators to remove excess periodate, quencher andthe byproducts thereof.

The retentate containing oxidized interferon-alpha is next mixed with anaqueous m-toluidine solution (50 mM) and incubated for 30 min at roomtemperature. Aminooxy-PEG reagent with a MW of 20 kD is then added togive a 5-fold molar reagent excess. This mixture is incubated for 2.5 hat room temperature in the dark under gentle stirring.

Finally, the PEG-interferon-alpha conjugate is purified by ion-exchangechromatography (e.g., on Q-Sepharose FF). For example, 1.5 mg protein/mlgel is loaded on the column equilibrated with 50 mM Hepes buffer, pH 7.4containing 5 mM CaCl₂. The conjugate is eluted with 50 mM Hepes buffercontaining 5 mM CaCl₂ and 500 mM sodium chloride, pH 7.4 and is thensubjected to UF/DF using an appropriate MW cutoff membrane. Thepreparation is next analytically characterized by measuring totalprotein (Coomassie, Bradford) and biological activity according tomethods known in the art.

In an alternative embodiment, Method 1 is carried out as follows.Interferon-alpha is PEGylated by use of a linear 20 kD PEGylationreagent containing an aminooxy group. An example of this type of reagentis the Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan).Interferon-alpha is dissolved in 7.0 ml histidine buffer, pH 6.0 (20 mML-histidine, 150 mM NaCl, 5 mM CaCl₂). An aqueous sodium periodatesolution (5 mM) is then added and the reaction mixture is incubated for1 h in the dark at 4° C. under gentle stirring and quenched for 15 minat room temperature by the addition of 7.5 μl of a 1 M aqueous cysteinesolution. The mixture is subsequently subjected to UF/DF employingVivaspin centrifugal filtrators to remove excess periodate, quencher andthe byproducts thereof.

The retentate containing oxidized interferon-alpha is next mixed with anaqueous m-toluidine solution (50 mM) and incubated for 30 min at roomtemperature. Aminooxy-PEG reagent with a MW of 20 kD is then added togive a 5-fold molar reagent excess. This mixture is incubated for 2.5 hat room temperature in the dark under gentle stirring.

Finally, the PEG-interferon-alpha conjugate is purified by ion-exchangechromatography. The conjugate containing fractions are collected andthen subjected to UF/DF using an appropriate MW cutoff membrane. Thepreparation is next analytically characterized by measuring totalprotein (Coomassie, Bradford) and biological activity according tomethods known in the art.

Method 2:

Interferon-alpha is PEGylated by use of a linear 20 kD PEGylationreagent containing an aminooxy group. An example of this type of reagentis the Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan).Interferon-alpha is transferred or dissolved in reaction buffer (e.g. 50mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to geta final protein concentration of 1.0+/−0.25 mg/ml. Then the pH of thesolution is corrected to 6.0 by drop wise addition of a 0.5 N aqueousHCl solution. Subsequently, a 40 mM aqueous sodium periodate solution isadded within 10 minutes to give a concentration of 200 μM. The oxidationreaction is carried out for 30+/−5 min at a temperature (T) ofT=+22+/−2° C. Then the reaction is stopped by addition of an aqueousL-cysteine solution (1 M) within 15 minutes at T=+22+/−2° C. to give afinal concentration of 10 mM in the reaction mixture and incubation for60+/−5 min.

The oxidized interferon-alpha is further purified by ion exchangechromatography. The oxidized interferon-alpha containing fractions ofthe eluate are collected and used for the conjugation reaction.

The aminooxy-PEG reagent with a MW of 20 kD reagent is added in a50-fold molar excess to the eluate containing the purified oxidizedinterferon-alpha within a maximum time period (t) of 15 minutes undergentle stirring. Then an aqueous m-toluidine solution (50 mM) is addedwithin 15 minutes to get a final concentration of 10 mM. The reactionmixture is incubated for 120+/−10 min. in the dark at a temperature (T)of T=+22+/−2° C. under gentle shaking.

The obtained PEG-interferon-alpha conjugate is further purified by ionexchange chromatography. The PEG-interferon alpha conjugate containingfractions are collected and concentrated by ultra-/diafiltration (UF/DF)using a membrane made of regenerated cellulose with an appropriatemolecular weight cut off (Millipore).

The conjugate prepared by use of this procedure are analyticallycharacterized by measuring total protein and biological activityaccording to methods known in the art.

Method 3:

Interferon-alpha is PEGylated by use of a linear 20 kD PEGylationreagent containing an aminooxy group. An example of this type of reagentis the Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan).Interferon-alpha is dissolved in Hepes buffer (50 mM Hepes, 150 mMsodium chloride, 5 mM calcium chloride, pH 6.0) and mixed with anaqueous sodium periodate solution (10 mM), and an aqueous m-toluidinesolution (50 mM). Subsequently the aminooxy reagent is added to give a20-fold molar reagent excess. The mixture is incubated for 2 h in thedark at room temperature under gentle stirring and quenched for 15 minat room temperature by the addition of 8 μl of aqueous cysteine solution(1 M).

Finally, the PEG-interferon-alpha conjugate is purified by ion-exchangechromatography on Q-Sepharose FF. 1.5 mg protein/ml gel is loaded on thecolumn pre equilibrated with 50 mM Hepes buffer, pH 7.4 containing 5 mMCaCl₂. The conjugate is eluted with 50 mM Hepes buffer containing 5 mMCaCl₂ and 500 mM sodium chloride, pH 7.4 and is then subjected to UF/DFusing a membrane. The preparation is analytically characterized bymeasuring total protein (Bradford) and biological activity according tomethods known in the art.

In an alternative embodiment, Method 3 is carried out as follows.Interferon-alpha is PEGylated by use of a linear 20 kD PEGylationreagent containing an aminooxy group. An example of this type of reagentis the Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan).Interferon-alpha is dissolved in Hepes buffer (50 mM Hepes, 150 mMsodium chloride, 5 mM calcium chloride, pH 6.0) and mixed with anaqueous sodium periodate solution (10 mM), and an aqueous m-toluidinesolution (50 mM). Subsequently the aminooxy reagent is added to give a20-fold molar reagent excess. The mixture is incubated for 2 h in thedark at room temperature under gentle stirring and quenched for 15 minat room temperature by the addition of 8 μl of aqueous cysteine solution(1 M).

Finally, the PEG-interferon-alpha conjugate is purified by ion-exchangechromatography. The conjugate containing fractions are collected andthen subjected to UF/DF using a membrane. The preparation isanalytically characterized by measuring total protein (Bradford) andbiological activity according to methods known in the art.

Method 4:

Interferon-alpha is PEGylated by use of a linear 20 kD PEGylationreagent containing an aminooxy group. An example of this type of reagentis the Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). Aninitial concentration or weight of interferon-alpha is transferred ordissolved in Hepes buffer (50 mM Hepes, 150 mM sodium chloride, 5 mMcalcium chloride, pH 6.0) to get a final protein concentration of 2 mginterferon-alpha/ml. Subsequently, an 5 mM aqueous sodium periodatesolution is added within 15 minutes to give a final concentration of 100μM, followed by addition of an 50 mM aqueous m-toluidine solution to geta final concentration of 10 mM within a time period of 30 minutes. Thenthe aminooxy-PEG reagent with a MW of 20 kD (described above) is addedto give a 20-fold molar reagent excess. After correction of the pH to6.0 the mixture is incubated for 2 h in the dark at room temperatureunder gentle stirring and quenched for 15 min at room temperature by theaddition of an 1 M aqueous L-cysteine solution to give a finalconcentration of 10 mM.

The PEG-interferon-alpha conjugate is purified by means of ion exchangechromatography (IEC). The conjugate containing fractions of the eluateare concentrated by UF/DF using a 10 kD membrane made of regeneratedcellulose (88 cm2, cut-off 10 kD/Millipore). The final diafiltrationstep is performed against Hepes buffer (50 mM Hepes, 5 mM CaCl₂, pH7.5).

The preparation is analytically characterized by measuring total protein(Bradford and BCA procedure) and biological activity according to knownmethods.

Example 41 PEGylation of Interferon-Gamma Using an Aminooxy-PEG Reagentand m-Toluidine as a Nucleophilic Catalyst

Method 1:

Interferon-gamma is PEGylated by use of a linear 20 kD PEGylationreagent containing an aminooxy group. An example of this type of reagentis the Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). 10 mgInterferon-gamma is dissolved in 5 ml histidine buffer, pH 6.0 (20 mML-histidine, 150 mM NaCl). 100 μl of an aqueous sodium periodatesolution (5 mM) is then added and the reaction mixture is incubated for1 h in the dark at 4° C. under gentle stirring and quenched for 15 minat room temperature by the addition of 50 μl of a 1 M aqueous cysteinesolution. The mixture is subsequently subjected to UF/DF employingVivaspin 15R 10 kD centrifugal filtrators to remove excess periodate,quencher and the byproducts thereof.

The retentate (approx. 7 ml), containing oxidized interferon-gamma, ismixed with 2 ml of an aqueous m-toluidine solution (50 mM) and incubatedfor 30 min at room temperature. Then aminooxy-PEG reagent with a MW of20 kD (described above) is added to give a 5-fold molar reagent excess.This mixture is incubated for 2.5 h at RT in the dark under gentlestirring.

Finally, the PEG-interferon-gamma conjugate is purified by ion-exchangechromatography on SP Sepharose FF. The reaction mixture is diluted with20 ml Buffer A (50 mM Hepes, pH 6.5) and loaded onto a 20 ml HiPrep SPFF16/10 column (GE Healthcare, Fairfield, Conn.) pre-equilibrated withBuffer A. Then the column is eluted with Buffer B (50 mM Hepes, 1 MNaCl, pH 6.5). Free interferon-gamma is eluted by washing the columnwith 25% Buffer B and the conjugate at 50% Buffer B. The conjugatecontaining fractions are concentrated by UF/DF using a 10 kD membranemade of regenerated cellulose (88 cm2, cut-off 10 kD/Millipore). Thefinal diafiltration step is performed against histidine buffer, pH 6.9containing 150 mM NaCl. The preparation is analytically characterized bymeasuring total protein (Bradford) and biological activity according tomethods known in the art. For the PEG-interferon-gamma conjugate aspecific activity of >50% in comparison to native Interferon gamma isdetermined. The conjugate is additionally analytically characterized bySize Exclusion HPLC using a Agilent 1200 HPLC system equipped with aShodex KW 803 column under conditions as previously described (Kolarichet al, Transfusion 2006; 46:1959-77). It is shown that the preparationcontains no free Interferon-gamma.

Method 2:

Interferon-gamma is PEGylated by use of a linear 20 kD PEGylationreagent containing an aminooxy group. An example of this type of reagentis the Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan).Interferon-gamma is transferred or dissolved in reaction buffer (e.g. 50mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to geta final protein concentration of 1.0+/−0.25 mg/ml. Then the pH of thesolution is corrected to 6.0 by drop wise addition of a 0.5 N aqueousHCl solution. Subsequently a 40 mM aqueous sodium periodate solution isadded within 10 minutes to give a concentration of 200 μM. The oxidationreaction is carried out for 30+/−5 min at a temperature (T) ofT=+22+/−2° C. Then the reaction is stopped by addition of an aqueousL-cysteine solution (1 M) within 15 minutes at T=+22+/−2° C. to give afinal concentration of 10 mM in the reaction mixture and incubation for60+/−5 min.

The oxidized interferon-gamma is further purified by ion exchangechromatography. The oxidized interferon-gamma containing fractions ofthe eluate are collected and used for the conjugation reaction.

The aminooxy-PEG reagent with a MW of 20 kD reagent is added in a50-fold molar excess to the eluate containing the purified oxidizedinterferon-gamma within a maximum time period (t) of 15 minutes undergentle stirring. Then an aqueous m-toluidine solution (50 mM) is addedwithin 15 minutes to get a final concentration of 10 mM. The reactionmixture is incubated for 120+/−10 min. in the dark at a temperature (T)of T=+22+/−2° C. under gentle shaking.

The obtained PEG-interferon-gamma conjugate is further purified by ionexchange chromatography. The PEG-interferon-gamma conjugate containingfractions are collected and concentrated by ultra-/diafiltration (UF/DF)using a membrane made of regenerated cellulose with an appropriatemolecular weight cut off (Millipore).

The conjugate prepared by use of this procedure are analyticallycharacterized by measuring total protein and biological activityaccording to methods known in the art.

Method 3:

Interferon-gamma is PEGylated by use of a linear 20 kD PEGylationreagent containing an aminooxy group. An example of this type of reagentis the Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). 10 mginterferon-gamma is dissolved in ˜8 ml histidine-buffer, pH 6.0 (20 mML-histidine, 150 mM NaCl). 200 μl of an aqueous sodium periodatesolution (5 mM) and 2 ml of an aqueous m-toluidine solution (50 mM) arethen added. Subsequently the aminooxy-PEG reagent with a MW of 20 kD(described above) is added to give a 5-fold molar reagent excess. Themixture is incubated for 2 h in the dark at room temperature undergentle stirring and quenched for 15 min at room temperature by theaddition of 100 μl of 1 M aqueous cysteine solution.

Finally the PEG-interferon-gamma conjugate is purified by ion-exchangechromatography on SP-Sepharose FF. The reaction mixture is diluted with20 ml Buffer A (50 mM Hepes, pH 6.5) and loaded onto a 20 ml HiPrep SPFF 16/10 column (GE Healthcare, Fairfield, Conn.) pre-equilibrated withBuffer A. Then the column is eluted with Buffer B (50 mM Hepes, 1 MNaCl, pH 6.5). Free interferon-gamma is eluted by washing the columnwith 25% Buffer B and the conjugate at 50% Buffer B. The conjugatecontaining fractions are concentrated by UF/DF using a 10 kD membranemade of regenerated cellulose (88 cm2, cut-off 10 kD/Millipore). Thefinal diafiltration step is performed against histidine buffer, pH 6.9containing 150 mM NaCl. The preparation is analytically characterized bymeasuring total protein (Bradford) and biological activity according tomethods known in the art. For the PEG-interferon-gamma conjugate aspecific activity of >50% in comparison to native interferon-gamma isdetermined. The conjugate is additionally analytically characterized bySize Exclusion HPLC using a Agilent 1200 HPLC system equipped with aShodex KW 803 column under conditions as previously described (Kolarichet al, Transfusion 2006; 46:1959-77). It is shown that the preparationcontains no free interferon-gamma.

Method 4:

Interferon-gamma is PEGylated by use of a linear 20 kD PEGylationreagent containing an aminooxy group. An example of this type of reagentis the Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). Aninitial concentration or weight of interferon-gamma is transferred ordissolved in Hepes buffer (50 mM Hepes, 150 mM sodium chloride, 5 mMcalcium chloride, pH 6.0) to get a final protein concentration of 2 mginterferon-gamma/ml. Subsequently an 5 mM aqueous sodium periodatesolution is added within 15 minutes to give a final concentration of 100μM, followed by addition of an 50 mM aqueous m-toluidine solution to geta final concentration of 10 mM within a time period of 30 minutes. Thenthe aminooxy-PEG reagent with a MW of 20 kD (described above) is addedto give a 20-fold molar reagent excess. After correction of the pH to6.0 the mixture is incubated for 2 h in the dark at room temperatureunder gentle stirring and quenched for 15 min at room temperature by theaddition of an 1 M aqueous L-cysteine solution to give a finalconcentration of 10 mM.

The PEG-interferon-gamma conjugate is purified by means of ion exchangechromatography (IEC). The conjugate containing fractions of the eluateare concentrated by UF/DF using a 10 kD membrane made of regeneratedcellulose (88 cm2, cut-off 10 kD/Millipore). The final diafiltrationstep is performed against Hepes buffer (50 mM Hepes, mM CaCl₂, pH 7.5).

The preparation is analytically characterized by measuring total protein(Bradford and BCA procedure) and biological activity according to knownmethods.

Example 42 PEGylation of G-CSF Using an Aminooxy-PEG Reagent andm-Toluidine as a Nucleophilic Catalyst

Method 1:

G-CSF is PEGylated by use of a linear 20 kD PEGylation reagentcontaining an aminooxy group. An example of this type of reagent is theSunbright® CA series from NOF (NOF Corp., Tokyo, Japan). G-CSF isdissolved in 7.0 ml histidine buffer, pH 6.0 (20 mM L-histidine, 150 mMNaCl, 5 mM CaCl₂). An aqueous sodium periodate solution (5 mM) is thenadded and the reaction mixture is incubated for 1 h in the dark at 4° C.under gentle stirring and quenched for 15 min at room temperature by theaddition of 7.5 μl of a 1 M aqueous cysteine solution. The mixture issubsequently subjected to UF/DF employing Vivaspin centrifugalfiltrators to remove excess periodate, quencher and the byproductsthereof.

The retentate containing oxidized G-CSF is next mixed with an aqueousm-toluidine solution (50 mM) and incubated for 30 min at roomtemperature. Aminooxy-PEG reagent with a MW of 20 kD is then added togive a 5-fold molar reagent excess. This mixture is incubated for 2.5 hat room temperature in the dark under gentle stirring.

Finally, the PEG-G-CSF conjugate is purified by ion-exchangechromatography (e.g., on Q-Sepharose FF). For example, 1.5 mg protein/mlgel is loaded on the column equilibrated with 50 mM Hepes buffer, pH 7.4containing 5 mM CaCl₂. The conjugate is eluted with 50 mM Hepes buffercontaining 5 mM CaCl₂ and 500 mM sodium chloride, pH 7.4 and is thensubjected to UF/DF using an appropriate MW cutoff membrane. Thepreparation is next analytically characterized by measuring totalprotein (Coomassie, Bradford) and biological activity according tomethods known in the art.

In an alternative embodiment, Method 1 is carried out as follows. G-CSFis PEGylated by use of a linear 20 kD PEGylation reagent containing anaminooxy group. An example of this type of reagent is the Sunbright® CAseries from NOF (NOF Corp., Tokyo, Japan). G-CSF is dissolved in 7.0 mlhistidine buffer, pH 6.0 (20 mM L-histidine, 150 mM NaCl, 5 mM CaCl₂).An aqueous sodium periodate solution (5 mM) is then added and thereaction mixture is incubated for 1 h in the dark at 4° C. under gentlestirring and quenched for 15 min at room temperature by the addition of7.5 μl of a 1 M aqueous cysteine solution. The mixture is subsequentlysubjected to UF/DF employing Vivaspin centrifugal filtrators to removeexcess periodate, quencher and the byproducts thereof.

The retentate containing oxidized G-CSF is next mixed with an aqueousm-toluidine solution (50 mM) and incubated for 30 min at roomtemperature. Aminooxy-PEG reagent with a MW of 20 kD is then added togive a 5-fold molar reagent excess. This mixture is incubated for 2.5 hat room temperature in the dark under gentle stirring.

Finally, the PEG-G-CSF conjugate is purified by ion-exchangechromatography (The conjugate containing fractions of the eluate arecollected and then subjected to UF/DF using an appropriate MW cutoffmembrane. The preparation is next analytically characterized bymeasuring total protein (Coomassie, Bradford) and biological activityaccording to methods known in the art.

Method 2:

G-CSF is PEGylated by use of a linear 20 kD PEGylation reagentcontaining an aminooxy group. An example of this type of reagent is theSunbright® CA series from NOF (NOF Corp., Tokyo, Japan). G-CSF istransferred or dissolved in reaction buffer (e.g. 50 mM Hepes, 350 mMsodium chloride, 5 mM calcium chloride, pH 6.0) to get a final proteinconcentration of 1.0+/−0.25 mg/ml. Then the pH of the solution iscorrected to 6.0 by drop wise addition of a 0.5N aqueous HCl solution.Subsequently a 40 mM aqueous sodium periodate solution is added within10 minutes to give a concentration of 200 μM. The oxidation reaction iscarried out for 30+/−5 min at a temperature (T) of T=+22+/−2° C. Thenthe reaction is stopped by addition of an aqueous L-cysteine solution (1M) within 15 minutes at T=+22+/−2° C. to give a final concentration of10 mM in the reaction mixture and incubation for 60+/−5 min.

The oxidized G-CSF is further purified by ion exchange chromatography.The oxidized G-CSF containing fractions of the eluate are collected andused for the conjugation reaction.

The aminooxy-PEG reagent with a MW of 20 kD reagent is added in a50-fold molar excess to the eluate containing the purified oxidizedG-CSF within a maximum time period (t) of 15 minutes under gentlestirring. Then an aqueous m-toluidine solution (50 mM) is added within15 minutes to get a final concentration of 10 mM. The reaction mixtureis incubated for 120+/−10 min. in the dark at a temperature (T) ofT=+22+/−2° C. under gentle shaking.

The obtained PEG-G-CSF conjugate is further purified by ion exchangechromatography. The PEG-G-CSF conjugate containing fractions arecollected and concentrated by ultra-/diafiltration (UF/DF) using amembrane made of regenerated cellulose with an appropriate molecularweight cut off (Millipore).

Method 3:

G-CSF is PEGylated by use of a linear 20 kD PEGylation reagentcontaining an aminooxy group. An example of this type of reagent is theSunbright® CA series from NOF (NOF Corp., Tokyo, Japan). G-CSF isdissolved in Hepes buffer (50 mM Hepes, 150 mM sodium chloride, 5 mMcalcium chloride, pH 6.0) and mixed with an aqueous sodium periodatesolution (10 mM), and an aqueous m-toluidine solution (50 mM).Subsequently, the aminooxy reagent is added to give a 20-fold molarreagent excess. The mixture is incubated for 2 h in the dark at roomtemperature under gentle stirring and quenched for 15 min at roomtemperature by the addition of 8 μl of aqueous cysteine solution (1 M).

Finally, the PEG-G-CSF conjugate is purified by ion-exchangechromatography on Q-Sepharose FF. 1.5 mg protein/ml gel is loaded on thecolumn pre equilibrated with 50 mM Hepes buffer, pH 7.4 containing 5 mMCaCl₂. The conjugate is eluted with 50 mM Hepes buffer containing 5 mMCaCl₂ and 500 mM sodium chloride, pH 7.4 and is then subjected to UF/DFusing a membrane. The preparation is analytically characterized bymeasuring total protein (Bradford) and biological activity according tomethods known in the art.

In an alternative embodiment, Method 3 is carried out as follows. G-CSFis PEGylated by use of a linear 20 kD PEGylation reagent containing anaminooxy group. An example of this type of reagent is the Sunbright® CAseries from NOF (NOF Corp., Tokyo, Japan). G-CSF is dissolved in Hepesbuffer (50 mM Hepes, 150 mM sodium chloride, 5 mM calcium chloride, pH6.0) and mixed with an aqueous sodium periodate solution (10 mM), and anaqueous m-toluidine solution (50 mM). Subsequently, the aminooxy reagentis added to give a 20-fold molar reagent excess. The mixture isincubated for 2 h in the dark at room temperature under gentle stirringand quenched for 15 min at room temperature by the addition of 8 μl ofaqueous cysteine solution (1 M).

Finally, the PEG-G-CSF conjugate is purified by ion-exchangechromatography. The conjugate containing fractions of the eluate arecollected and then subjected to UF/DF using a membrane. The preparationis analytically characterized by measuring total protein (Bradford) andbiological activity according to methods known in the art.

Method 4:

G-CSF is PEGylated by use of a linear 20 kD PEGylation reagentcontaining an aminooxy group. An example of this type of reagent is theSunbright® CA series from NOF (NOF Corp., Tokyo, Japan). An initialconcentration or weight of G-CSF is transferred or dissolved in Hepesbuffer (50 mM Hepes, 150 mM sodium chloride, 5 mM calcium chloride, pH6.0) to get a final protein concentration of 2 mg G-CSF/ml.Subsequently, an 5 mM aqueous sodium periodate solution is added within15 minutes to give a final concentration of 100 μM, followed by additionof an 50 mM aqueous m-toluidine solution to get a final concentration of10 mM within a time period of 30 minutes. Then the aminooxy-PEG reagentwith a MW of 20 kD (described above) is added to give a 20-fold molarreagent excess. After correction of the pH to 6.0 the mixture isincubated for 2 h in the dark at room temperature under gentle stirringand quenched for 15 min at room temperature by the addition of an 1 Maqueous L-cysteine solution to give a final concentration of 10 mM.

The G-CSF conjugate is purified by means of ion exchange chromatography(IEC). The conjugate containing fractions of the eluate are concentratedby UF/DF using a 10 kD membrane made of regenerated cellulose (88 cm2,cut-off 10 kD/Millipore). The final diafiltration step is performedagainst Hepes buffer (50 mM Hepes, 5 mM CaCl₂, pH 7.5).

The preparation is analytically characterized by measuring total protein(Bradford and BCA procedure) and biological activity according to knownmethods.

Example 43 PEGylation of Humira Using an Aminooxy-PEG Reagent andm-Toluidine as a Nucleophilic Catalyst

Method 1:

Humira is PEGylated by use of a linear 20 kD PEGylation reagentcontaining an aminooxy group. An example of this type of reagent is theSunbright® CA series from NOF (NOF Corp., Tokyo, Japan). Humira isdissolved in 7.0 ml histidine buffer, pH 6.0 (20 mM L-histidine, 150 mMNaCl, 5 mM CaCl₂). An aqueous sodium periodate solution (5 mM) is thenadded and the reaction mixture is incubated for 1 h in the dark at 4° C.under gentle stirring and quenched for 15 min at room temperature by theaddition of 7.5 μl of a 1 M aqueous cysteine solution. The mixture issubsequently subjected to UF/DF employing Vivaspin centrifugalfiltrators to remove excess periodate, quencher and the byproductsthereof.

The retentate containing oxidized Humira is next mixed with an aqueousm-toluidine solution (50 mM) and incubated for 30 min at roomtemperature. Aminooxy-PEG reagent with a MW of 20 kD is then added togive a 5-fold molar reagent excess. This mixture is incubated for 2.5 hat room temperature in the dark under gentle stirring.

Finally, the PEG-Humira conjugate is purified by ion-exchangechromatography (e.g., on Q-Sepharose FF). For example, 1.5 mg protein/mlgel is loaded on the column equilibrated with 50 mM Hepes buffer, pH 7.4containing 5 mM CaCl₂. The conjugate is eluted with 50 mM Hepes buffercontaining 5 mM CaCl₂ and 500 mM sodium chloride, pH 7.4 and is thensubjected to UF/DF using an appropriate MW cutoff membrane. Thepreparation is next analytically characterized by measuring totalprotein (Coomassie, Bradford) and biological activity according tomethods known in the art.

In an alternative embodiment, Method 1 is carried out as follows. Humirais PEGylated by use of a linear 20 kD PEGylation reagent containing anaminooxy group. An example of this type of reagent is the Sunbright® CAseries from NOF (NOF Corp., Tokyo, Japan). Humira is dissolved in 7.0 mlhistidine buffer, pH 6.0 (20 mM L-histidine, 150 mM NaCl, 5 mM CaCl₂).An aqueous sodium periodate solution (5 mM) is then added and thereaction mixture is incubated for 1 h in the dark at 4° C. under gentlestirring and quenched for 15 min at room temperature by the addition of7.5 μl of a 1 M aqueous cysteine solution. The mixture is subsequentlysubjected to UF/DF employing Vivaspin centrifugal filtrators to removeexcess periodate, quencher and the byproducts thereof.

The retentate containing oxidized Humira is next mixed with an aqueousm-toluidine solution (50 mM) and incubated for 30 min at roomtemperature. Aminooxy-PEG reagent with a MW of 20 kD is then added togive a 5-fold molar reagent excess. This mixture is incubated for 2.5 hat room temperature in the dark under gentle stirring.

Finally, the PEG-Humira conjugate is purified by ion-exchangechromatography. The conjugate containing fractions of the eluate arecollected and then subjected to UF/DF using an appropriate MW cutoffmembrane. The preparation is next analytically characterized bymeasuring total protein (Coomassie, Bradford) and biological activityaccording to methods known in the art.

Method 2:

Humira is PEGylated by use of a linear 20 kD PEGylation reagentcontaining an aminooxy group. An example of this type of reagent is theSunbright® CA series from NOF (NOF Corp., Tokyo, Japan). Humira istransferred or dissolved in reaction buffer (e.g. 50 mM Hepes, 350 mMsodium chloride, 5 mM calcium chloride, pH 6.0) to get a final proteinconcentration of 1.0+/−0.25 mg/ml. Then the pH of the solution iscorrected to 6.0 by drop wise addition of a 0.5N aqueous HCl solution.Subsequently a 40 mM aqueous sodium periodate solution is added within10 minutes to give a concentration of 200 μM. The oxidation reaction iscarried out for 30+/−5 min at a temperature (T) of T=+22+/−2° C. Thenthe reaction is stopped by addition of an aqueous L-cysteine solution (1M) within 15 minutes at T=+22+/−2° C. to give a final concentration of10 mM in the reaction mixture and incubation for 60+/−5 min.

The oxidized Humira is further purified by ion exchange chromatography.The oxidized Humira containing fractions of the eluate are collected andused for the conjugation reaction.

The aminooxy-PEG reagent with a MW of 20 kD reagent is added in a50-fold molar excess to the eluate containing the purified oxidizedHumira within a maximum time period (t) of 15 minutes under gentlestirring. Then an aqueous m-toluidine solution (50 mM) is added within15 minutes to get a final concentration of 10 mM. The reaction mixtureis incubated for 120+/−10 min. in the dark at a temperature (T) ofT=+22+/−2° C. under gentle shaking.

The obtained PEG-Humira conjugate is further purified by ion exchangechromatography. The PEG-Humira conjugate containing fractions arecollected and concentrated by ultra-/diafiltration (UF/DF) using amembrane made of regenerated cellulose with an appropriate molecularweight cut off (Millipore).

Method 3:

Humira is PEGylated by use of a linear 20 kD PEGylation reagentcontaining an aminooxy group. An example of this type of reagent is theSunbright® CA series from NOF (NOF Corp., Tokyo, Japan). Humira isdissolved in Hepes buffer (50 mM Hepes, 150 mM sodium chloride, 5 mMcalcium chloride, pH 6.0) and mixed with an aqueous sodium periodatesolution (10 mM), and an aqueous m-toluidine solution (50 mM).Subsequently, the aminooxy reagent is added to give a 20-fold molarreagent excess. The mixture is incubated for 2 h in the dark at roomtemperature under gentle stirring and quenched for 15 min at roomtemperature by the addition of 8 μl of aqueous cysteine solution (1 M).

Finally, the PEG-Humira conjugate is purified by ion-exchangechromatography on Q-Sepharose FF. 1.5 mg protein/ml gel is loaded on thecolumn pre equilibrated with 50 mM Hepes buffer, pH 7.4 containing 5 mMCaCl₂. The conjugate is eluted with 50 mM Hepes buffer containing 5 mMCaCl₂ and 500 mM sodium chloride, pH 7.4 and is then subjected to UF/DFusing a membrane. The preparation is analytically characterized bymeasuring total protein (Bradford) and biological activity according tomethods known in the art.

In an alternative embodiment, Method 3 is carried out as follows. Humirais PEGylated by use of a linear 20 kD PEGylation reagent containing anaminooxy group. An example of this type of reagent is the Sunbright® CAseries from NOF (NOF Corp., Tokyo, Japan). Humira is dissolved in Hepesbuffer (50 mM Hepes, 150 mM sodium chloride, 5 mM calcium chloride, pH6.0) and mixed with an aqueous sodium periodate solution (10 mM), and anaqueous m-toluidine solution (50 mM). Subsequently the aminooxy reagentis added to give a 20-fold molar reagent excess. The mixture isincubated for 2 h in the dark at room temperature under gentle stirringand quenched for 15 min at room temperature by the addition of 8 μl ofaqueous cysteine solution (1 M).

Finally, the PEG-Humira conjugate is purified by ion-exchangechromatography. The conjugate containing fractions are collected andthen subjected to UF/DF using a membrane. The preparation isanalytically characterized by measuring total protein (Bradford) andbiological activity according to methods known in the art.

Method 4:

Humira is PEGylated by use of a linear 20 kD PEGylation reagentcontaining an aminooxy group. An example of this type of reagent is theSunbright® CA series from NOF (NOF Corp., Tokyo, Japan). An initialconcentration or weight of Humira is transferred or dissolved in Hepesbuffer (50 mM Hepes, 150 mM sodium chloride, 5 mM calcium chloride, pH6.0) to get a final protein concentration of 2 mg Humira/ml.Subsequently an 5 mM aqueous sodium periodate solution is added within15 minutes to give a final concentration of 100 μM, followed by additionof an 50 mM aqueous m-toluidine solution to get a final concentration of10 mM within a time period of 30 minutes. Then the aminooxy-PEG reagentwith a MW of 20 kD (described above) is added to give a 20-fold molarreagent excess. After correction of the pH to 6.0 the mixture isincubated for 2 h in the dark at room temperature under gentle stirringand quenched for 15 min at room temperature by the addition of a 1 Maqueous L-cysteine solution to give a final concentration of 10 mM.

The Humira conjugate is purified by means of ion exchange chromatography(IEC). The conjugate containing fractions of the eluate are concentratedby UF/DF using a 10 kD membrane made of regenerated cellulose (88 cm2,cut-off 10 kD/Millipore). The final diafiltration step is performedagainst Hepes buffer (50 mM Hepes, 5 mM CaCl₂, pH 7.5).

The preparation is analytically characterized by measuring total protein(Bradford and BCA procedure) and biological activity according to knownmethods.

Example 44 PEGylation of Prolia Using an Aminooxy-PEG Reagent andm-Toluidine as a Nucleophilic Catalyst

Method 1:

Prolia is PEGylated by use of a linear 20 kD PEGylation reagentcontaining an aminooxy group. An example of this type of reagent is theSunbright® CA series from NOF (NOF Corp., Tokyo, Japan). Prolia isdissolved in 7.0 ml histidine buffer, pH 6.0 (20 mM L-histidine, 150 mMNaCl, 5 mM CaCl₂). An aqueous sodium periodate solution (5 mM) is thenadded and the reaction mixture is incubated for 1 h in the dark at 4° C.under gentle stirring and quenched for 15 min at room temperature by theaddition of 7.5 μl of a 1 M aqueous cysteine solution. The mixture issubsequently subjected to UF/DF employing Vivaspin centrifugalfiltrators to remove excess periodate, quencher and the byproductsthereof.

The retentate containing oxidized Prolia is next mixed with an aqueousm-toluidine solution (50 mM) and incubated for 30 min at roomtemperature. Aminooxy-PEG reagent with a MW of 20 kD is then added togive a 5-fold molar reagent excess. This mixture is incubated for 2.5 hat room temperature in the dark under gentle stirring.

Finally, the PEG-Prolia conjugate is purified by ion-exchangechromatography (e.g., on Q-Sepharose FF). For example, 1.5 mg protein/mlgel is loaded on the column equilibrated with 50 mM Hepes buffer, pH 7.4containing 5 mM CaCl₂. The conjugate is eluted with 50 mM Hepes buffercontaining 5 mM CaCl₂ and 500 mM sodium chloride, pH 7.4 and is thensubjected to UF/DF using an appropriate MW cutoff membrane. Thepreparation is next analytically characterized by measuring totalprotein (Coomassie, Bradford) and biological activity according tomethods known in the art.

In an alternative embodiment, Method 1 is carried out as follows. Proliais PEGylated by use of a linear 20 kD PEGylation reagent containing anaminooxy group. An example of this type of reagent is the Sunbright® CAseries from NOF (NOF Corp., Tokyo, Japan). 10 mg rFIX is dissolved in 5ml histidine-buffer, pH 6.0 (20 mM L-histidine, 150 mM NaCl). 100 μl ofan aqueous sodium periodate solution (5 mM) is then added and thereaction mixture is incubated for 1 h in the dark at 4° C. under gentlestirring and quenched for 15 min at room temperature by the addition of50 μl of a 1 M aqueous cysteine solution. The mixture is subsequentlysubjected to UF/DF employing Vivaspin 15R 10 kD centrifugal filtratorsto remove excess periodate, quencher and the byproducts thereof.

The retentate (approx. 7 ml), containing oxidized Prolia, is mixed with2 ml of an aqueous m-toluidine solution (50 mM) and incubated for 30 minat room temperature. Then aminooxy-PEG reagent with a MW of 20 kD(described above) is added to give a 5-fold molar reagent excess. Thismixture is incubated for 2.5 h at RT in the dark under gentle stirring.

Finally the PEG-Prolia conjugate is purified by ion-exchangechromatography on SP Sepharose FF. The reaction mixture is diluted with20 ml Buffer A (50 mM Hepes, pH 6.5) and loaded onto a 20 ml HiPrep SPFF 16/10 column (GE Healthcare, Fairfield, Conn.) pre-equilibrated withBuffer A. Then the column is eluted with Buffer B (50 mM Hepes, 1 MNaCl, pH 6.5). Free Prolia is eluted by washing the column with 25%Buffer B and the conjugate at 50% Buffer B. The conjugate containingfractions are concentrated by UF/DF using a 10 kD membrane made ofregenerated cellulose (88 cm2, cut-off 10 kD/Millipore). The finaldiafiltration step is performed against histidine buffer, pH 6.9containing 150 mM NaCl. The preparation is analytically characterized bymeasuring total protein (Bradford) and biological activity according tomethods known in the art. For the PEG-Prolia conjugate a specificactivity of >50% in comparison to native Prolia is determined. Theconjugate is additionally analytically characterized by Size ExclusionHPLC using a Agilent 1200 HPLC system equipped with a Shodex KW 803column under conditions as previously described (Kolarich et al,Transfusion 2006; 46:1959-77). It is shown that the preparation containsno free Prolia.

Method 2:

Prolia is PEGylated by use of a linear 20 kD PEGylation reagentcontaining an aminooxy group. An example of this type of reagent is theSunbright® CA series from NOF (NOF Corp., Tokyo, Japan). Prolia istransferred or dissolved in reaction buffer (e.g. 50 mM Hepes, 350 mMsodium chloride, 5 mM calcium chloride, pH 6.0) to get a final proteinconcentration of 1.0+/−0.25 mg/ml. Then the pH of the solution iscorrected to 6.0 by drop wise addition of a 0.5N aqueous HCl solution.Subsequently, a 40 mM aqueous sodium periodate solution is added within10 minutes to give a concentration of 200 μM. The oxidation reaction iscarried out for 30+/−5 min at a temperature (T) of T=+22+/−2° C. Thenthe reaction is stopped by addition of an aqueous L-cysteine solution (1M) within 15 minutes at T=+22+/−2° C. to give a final concentration of10 mM in the reaction mixture and incubation for 60+/−5 min.

The oxidized Prolia is further purified by ion exchange chromatography.The oxidized Humira containing fractions of the eluate are collected andused for the conjugation reaction.

The aminooxy-PEG reagent with a MW of 20 kD reagent is added in a50-fold molar excess to the eluate containing the purified oxidizedProlia within a maximum time period (t) of 15 minutes under gentlestirring. Then an aqueous m-toluidine solution (50 mM) is added within15 minutes to get a final concentration of 10 mM. The reaction mixtureis incubated for 120+/−10 min. in the dark at a temperature (T) ofT=+22+/−2° C. under gentle shaking.

The obtained PEG-Prolia conjugate is further purified by ion exchangechromatography. The PEG-Prolia conjugate containing fractions arecollected and concentrated by ultra-/diafiltration (UF/DF) using amembrane made of regenerated cellulose with an appropriate molecularweight cut off (Millipore).

Method 3:

Prolia is PEGylated by use of a linear 20 kD PEGylation reagentcontaining an aminooxy group. An example of this type of reagent is theSunbright® CA series from NOF (NOF Corp., Tokyo, Japan). EPO isdissolved in Hepes buffer (50 mM Hepes, 150 mM sodium chloride, 5 mMcalcium chloride, pH 6.0) and mixed with an aqueous sodium periodatesolution (10 mM), and an aqueous m-toluidine solution (50 mM).Subsequently the aminooxy reagent is added to give a 20-fold molarreagent excess. The mixture is incubated for 2 h in the dark at roomtemperature under gentle stirring and quenched for 15 min at roomtemperature by the addition of 8 μl of aqueous cysteine solution (1 M).

Finally, the PEG-Prolia conjugate is purified by ion-exchangechromatography on Q-Sepharose FF. 1.5 mg protein/ml gel is loaded on thecolumn pre equilibrated with 50 mM Hepes buffer, pH 7.4 containing 5 mMCaCl₂. The conjugate is eluted with 50 mM Hepes buffer containing 5 mMCaCl₂ and 500 mM sodium chloride, pH 7.4 and is then subjected to UF/DFusing a membrane. The preparation is analytically characterized bymeasuring total protein (Bradford) and biological activity according tomethods known in the art.

In an alternative embodiment, Method 3 is carried out as follows.

Prolia is PEGylated by use of a linear 20 kD PEGylation reagentcontaining an aminooxy group. An example of this type of reagent is theSunbright® CA series from NOF (NOF Corp., Tokyo, Japan). 10 mg Prolia isdissolved in ˜8 ml histidine buffer, pH 6.0 (20 mM L-histidine, 150 mMNaCl). 200 μl of an aqueous sodium periodate solution (5 mM) and 2 ml ofan aqueous m-toluidine solution (50 mM) are then added. Subsequently,the aminooxy-PEG reagent with a MW of 20 kD (described above) is addedto give a 5-fold molar reagent excess. The mixture is incubated for 2 hin the dark at room temperature under gentle stirring and quenched for15 min at room temperature by the addition of 100 μl of 1 M aqueouscysteine solution.

Finally the PEG-Prolia conjugate is purified by ion-exchangechromatography on SP-Sepharose FF. The reaction mixture is diluted with20 ml Buffer A (50 mM Hepes, pH 6.5) and loaded onto a 20 ml HiPrep SPFF16/10 column (GE Healthcare, Fairfield, Conn.) pre-equilibrated withBuffer A. Then the column is eluted with Buffer B (50 mM Hepes, 1 MNaCl, pH 6.5). Free Prolia is eluted by washing the column with 25%Buffer B and the conjugate at 50% Buffer B. The conjugate containingfractions are concentrated by UF/DF using a 10 kD membrane made ofregenerated cellulose (88 cm2, cut-off 10 kD/Millipore). The finaldiafiltration step is performed against histidine buffer, pH 6.9containing 150 mM NaCl. The preparation is analytically characterized bymeasuring total protein (Bradford) and biological activity according tomethods known in the art. For the PEG-Prolia conjugate a specificactivity of >50% in comparison to native Prolia is determined. Theconjugate is additionally analytically characterized by Size ExclusionHPLC using a Agilent 1200 HPLC system equipped with a Shodex KW 803column under conditions as previously described (Kolarich et al,Transfusion 2006; 46:1959-77). It is shown that the preparation containsno free Prolia.

Method 4:

Prolia is PEGylated by use of a linear 20 kD PEGylation reagentcontaining an aminooxy group. An example of this type of reagent is theSunbright® CA series from NOF (NOF Corp., Tokyo, Japan). An initialconcentration or weight of Humira is transferred or dissolved in Hepesbuffer (50 mM Hepes, 150 mM sodium chloride, 5 mM calcium chloride, pH6.0) to get a final protein concentration of 2 mg Prolia/ml.Subsequently an 5 mM aqueous sodium periodate solution is added within15 minutes to give a final concentration of 100 μM, followed by additionof an 50 mM aqueous m-toluidine solution to get a final concentration of10 mM within a time period of 30 minutes. Then the aminooxy-PEG reagentwith a MW of 20 kD (described above) is added to give a 20-fold molarreagent excess. After correction of the pH to 6.0 the mixture isincubated for 2 h in the dark at room temperature under gentle stirringand quenched for 15 min at room temperature by the addition of an 1 Maqueous L-cysteine solution to give a final concentration of 10 mM.

The Prolia conjugate is purified by means of ion exchange chromatography(IEC). The conjugate containing fractions of the eluate are concentratedby UF/DF using a 10 kD membrane made of regenerated cellulose (88 cm2,cut-off 10 kD/Millipore). The final diafiltration step is performedagainst Hepes buffer (50 mM Hepes, 5 mM CaCl₂, pH 7.5).

The preparation is analytically characterized by measuring total protein(Bradford and BCA procedure) and biological activity according to knownmethods.

Example 45 PEGylation of a Therapeutic Protein Using Branched PEG

PEGylation of a therapeutic protein of the invention may be extended toa branched or linear PEGylation reagent, which is made of an aldehydeand a suitable linker containing an active aminooxy group.

Example 46 Coupling of a Diamonooxy Linker to Native PSA

This Example describes procedures to prepare aminooxy-PSA reagents usingnative PSA (i.e. without prior oxidation), which can be used forchemical modification of therapeutic proteins.

a) Coupling at Ambient Temperature

52.2 mg of native PSA (MW=20 kD) obtained from the Serum Institute ofIndia (Pune, India) was dissolved in 1.05 ml 50 mM phosphate buffer pH6.0. Then 10.3 mg 3-oxa-pentane-1,5-dioxyamine (linker molecule) wasadded drop wise to the reaction mixture. The reaction was incubated for2 hrs at room temperature under gentle agitation in the dark.

B) Coupling at Increased Temperature

52.2 mg of native PSA (MW=20 kD) obtained from the Serum Institute ofIndia (Pune, India) was dissolved in 1.05 ml 50 mM phosphate buffer pH6.0. Then 10.3 mg 3-oxa-pentane-1,5-dioxyamine (linker molecule) wasadded drop wise to the reaction mixture. The reaction was incubated for2 hrs at room temperature under gentle agitation in the dark. Then thetemperature was increased to 32-37° C. and the reaction mixture wasincubated for another 14 hrs.

C) Coupling at Increased Temperature and Increased Linker Excess

52.2 mg of native PSA (MW=20 kD) obtained from the Serum Institute ofIndia (Pune, India) was dissolved in 1.05 ml 50 mM phosphate buffer pH6.0. Then 10.3 mg 3-oxa-pentane-1,5-dioxyamine (linker molecule) wasadded drop wise to the reaction mixture. The reaction was incubated for2 hrs at room temperature under gentle agitation in the dark. Then 26.3mg 3-oxa-pentane-1,5-dioxyamine were added drop wise to the reaction,the temperature was increased to 32-37° C. and the reaction mixture wasincubated for another 14 hrs.

D) Purification of PSA Derivatives

After the incubation was completed, the reaction mixtures generatedunder points a-c were purified by extensive dialysis. Therefore samplesof the reaction mixtures were loaded into Slide-A-Lyzer dialysiscassettes (0-5-3 ml, MWCO 3.5 kD, reg. cellulose, Pierce) and dialyzedagainst 10 mM phosphate buffer pH 8.0 according to the followingpattern:

2 hrs against 500 ml buffer at room temperature

2 hrs against 500 ml buffer at room temperature

12 hrs against 500 ml buffer at 4° C.

1 h against 50 ml ‘Slide-A-Lyzer Concentrating Solution for Dialysis’ atroom temperature for concentration to initial sample volume.

The purified aminooxy-PSA is thus ready to be used in a proteinconjugation reaction according to, for example, Examples 11, 12, 14, and17-31, above. Likewise, any of the water-soluble polymers describedherein can be coupled to an aminooxy linker as described in this Exampleand then conjugated to a protein as set out in the above Examples.

The preparation was analytically characterized by measuring total PSA(Resorcinol assay) and total aminooxy groups (TNBS assay) to determinethe degree of modification. For preparation (a) a modification degree(MD) of 0.35, for (b) MD=0.54 and for (c) MD=0.58 was determined.Furthermore the polydispersity as well as free3-oxa-pentane-1,5-dioxyamine was measured. The polydispersity was lowerthan 1.15 for all preparations and the content of free linker was lowerthan 0.15 mol % of the PSA concentration.

For the PSA modified at the reducing end the following structure wasdetermined by ¹³C NMR spectroscopy.

Example 47 Preparation of Aminooxy-PSA at 4° C. Employing aChromatographic Purification Step

During a detailed analytical characterization of the aminooxy-PSAreagent prepared at room temperature, NMR studies (See, e.g., U.S.Provisional Application No. 61/647,814, incorporated by reference init's entirety) revealed that the derivatization of oxidized PSA with thediaminooxy linker consists of two distinct reactions: a quick reactionof the aldehyde group at the non-reducing end of PSA and a slow reactionof the aldehyde group (in the form of a hemiketal) at the reducing endof PSA. The latter reaction could be considered an unwanted sidereaction that is to be avoided for reagent production.

Therefore, the process for the preparation of the aminooxy-PSA reagenthas been optimized as described in the instant Example. The reducing endonly occurs to a significant degree if the process is performed at roomtemperature. Hence, the process was adjusted and is conducted at 2-8° C.By performing the whole process (chemical reaction and purification ofthe PSA reagent by IEX) at 2-8° C., the side reaction at the reducingend of PSA was substantially reduced. This process change thus leads toa reagent of higher quality.

Procedure

1290 mg of oxidized PSA (MW=20 kD) obtained from the Serum Institute ofIndia (Pune, India) was dissolved in 25 ml 50 mM phosphate buffer pH 6.0(Buffer A). Then 209 mg 3-oxa-pentane-1,5-dioxyamine was added to thereaction mixture and incubated for 1 h at 2-8° C. under gentle agitationin the dark.

After incubation, the mixture was subjected to a weak anion exchangechromatography step employing a Fractogel EMD DEAE 650-M chromatographygel (column dimension: XK26/135) carried out in a cold room attemperature of 2-8° C. The reaction mixture was diluted with pre-cooled110 ml Buffer A and loaded onto the DEAE column pre-equilibrated withBuffer A at a flow rate of 1 cm/min. Then the column was washed with 20CV Buffer B (20 mM Hepes, pH 6.0) at a flow rate of 2 cm/min to removefree 3-oxa-pentane-1,5-dioxyamine. The aminooxy-PSA reagent was theneluted with a step gradient consisting of 67% Buffer B and 43% Buffer C(20 mM Hepes, 1M NaCl, pH 7.5). The eluate was concentrated by UF/DFusing a 5 kD membrane made of polyether sulfone (50 cm2, Millipore). Thepreparation was analytically characterized by measuring total PSA(Resorcinol assay) and total aminooxy groups (TNBS assay) to determinethe degree of modification. The PSA concentration in the finalpreparation was 46.0 mg/ml and the modification degree was 83.5%.Furthermore a polydispersity value of 1.131 was determined. In additiona concentration of 0.22 μg/ml (0.07 mol % of PSA) was measured for free3-oxa-pentane-1,5-dioxyamine.

The purified aminooxy-PSA is thus ready to be used in a conjugationreaction according to Examples 11, 12, 14, and 17-31, above.

What is claimed is:
 1. A method of conjugating a water soluble polymerto an oxidized carbohydrate moiety of a therapeutic protein comprisingcontacting the oxidized carbohydrate moiety with an activated watersoluble polymer under conditions that allow conjugation; said watersoluble polymer containing an active aminooxy group and is selected fromthe group consisting of polyethylene glycol (PEG), branched PEG,polysialic acid (PSA), starch, hydroxyalkyl starch (HAS), hydroxylethylstarch (HES), carbohydrate, polysaccharides, pullulane, chitosan,hyaluronic acid, chondroitin sulfate, dermatan sulfate, starch, dextran,carboxymethyl-dextran, polyalkylene oxide (PAO), polyalkylene glycol(PAG), polypropylene glycol (PPG), polyoxazoline,polyacryloylmorpholine, polyvinyl alcohol (PVA), polycarboxylate,polyvinylpyrrolidone, polyphosphazene, polyoxazoline,polyethylene-co-maleic acid anhydride, polystyrene-co-maleic acidanhydride, poly(1hydroxymethylethylene hydroxymethylformal) (PHF),2-methacryloyloxy-2′-ethyltrimethylammoniumphosphate (MPC); wherein thewater soluble polymer containing an active aminooxy group is prepared bya method comprising: a) incubating a solution comprising an oxidizedwater-soluble polymer with an aminooxy linker comprising an activeaminooxy group under conditions that allow the formation of a stableoxime linkage between the oxidized water-soluble polymer and theactivated aminooxy linker, said conditions comprising a time periodbetween about 1 minute and about 24 hours; a temperature between about2° C. and about 10° C.; in the presence or absence of light, and with orwithout stirring; thereby forming a water-soluble polymer containing anactive aminooxy group; and b) purifying the water soluble polymercontaining an active aminooxy group by a method selected from the groupconsisting of chromatography, filtration, dialysis, and precipitation orcombinations thereof, at a temperature between about 2° C. and about 8°C.; said carbohydrate moiety oxidized by incubation with a buffercomprising an oxidizing agent selected from the group consisting ofsodium periodate (NaIO₄), and lead tetraacetate (Pb(OAc)₄); wherein anoxime linkage is formed between the oxidized carbohydrate moiety and theactive aminooxy group on the water soluble polymer; and wherein saidoxime linkage formation is catalyzed by m-toluidine.
 2. The methodaccording to claim 1 wherein the solution comprising the oxidizedwater-soluble polymer and the aminooxy linker comprising an activeaminooxy group is incubated at 4° C. for 1 h in the absence of lightwith stirring.
 3. The method according to claim 2 wherein the watersoluble polymer containing an active aminooxy group is purified by anionexchange chromatography at a temperature of 4° C.
 4. The methodaccording to claim 2 wherein the oxidized water soluble polymer is PSAand is oxidized by incubation with NaIO₄.
 5. The method according toclaim 1 wherein the therapeutic protein selected from the groupconsisting of Factor IX (FIX), Factor VIII (FVIII), Factor VIIa (FVIIa),Von Willebrand Factor (VWF), Factor FV (FV), Factor X (FX), Factor XI(FXI), Factor XII (FXII), thrombin (FII), protein C, protein S, tPA,PAI-1, tissue factor (TF), ADAMTS 13 protease, IL-1 alpha, IL-1 beta,IL-2, IL-3, IL-4, IL-5, IL-6, IL-11, colony stimulating factor-1(CSF-1), M-CSF, SCF, GM-CSF, granulocyte colony stimulating factor(G-CSF), EPO, interferon-alpha (IFN-alpha), consensus interferon,IFN-beta, IFN-gamma, IFN-omega, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13,IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23,IL-24, IL-31, IL-32 alpha, IL-33, thrombopoietin (TPO), Ang-1, Ang-2,Ang-4, Ang-Y, angiopoietin-like polypeptide 1 (ANGPTL1),angiopoietin-like polypeptide 2 (ANGPTL2), angiopoietin-like polypeptide3 (ANGPTL3), angiopoietin-like polypeptide 4 (ANGPTL4),angiopoietin-like polypeptide 5 (ANGPTL5), angiopoietin-like polypeptide6 (ANGPTL6), angiopoietin-like polypeptide 7 (ANGPTL7), vitronectin,vascular endothelial growth factor (VEGF), angiogenin, activin A,activin B, activin C, bone morphogenic protein-1, bone morphogenicprotein-2, bone morphogenic protein-3, bone morphogenic protein-4, bonemorphogenic protein-5, bone morphogenic protein-6, bone morphogenicprotein-7, bone morphogenic protein-8, bone morphogenic protein-9, bonemorphogenic protein-10, bone morphogenic protein-11, bone morphogenicprotein-12, bone morphogenic protein-13, bone morphogenic protein-14,bone morphogenic protein-15, bone morphogenic protein receptor IA, bonemorphogenic protein receptor IB, bone morphogenic protein receptor II,brain derived neurotrophic factor, cardiotrophin-1, ciliary neutrophicfactor, ciliary neutrophic factor receptor, cripto, cryptic,cytokine-induced neutrophil chemotactic factor 1, cytokine-inducedneutrophil, chemotactic factor 2α, cytokine-induced neutrophilchemotactic factor 2β, β endothelial cell growth factor, endothelin 1,epidermal growth factor, epigen, epiregulin, epithelial-derivedneutrophil attractant, fibroblast growth factor 4, fibroblast growthfactor 5, fibroblast growth factor 6, fibroblast growth factor 7,fibroblast growth factor 8, fibroblast growth factor 8b, fibroblastgrowth factor 8c, fibroblast growth factor 9, fibroblast growth factor10, fibroblast growth factor 11, fibroblast growth factor 12, fibroblastgrowth factor 13, fibroblast growth factor 16, fibroblast growth factor17, fibroblast growth factor 19, fibroblast growth factor 20, fibroblastgrowth factor 21, fibroblast growth factor acidic, fibroblast growthfactor basic, glial cell line-derived neutrophic factor receptor α1,glial cell line-derived neutrophic factor receptor α2, growth relatedprotein, growth related protein α, growth related protein β, growthrelated protein γ, heparin binding epidermal growth factor, hepatocytegrowth factor, hepatocyte growth factor receptor, hepatoma-derivedgrowth factor, insulin-like growth factor I, insulin-like growth factorreceptor, insulin-like growth factor II, insulin-like growth factorbinding protein, keratinocyte growth factor, leukemia inhibitory factor,leukemia inhibitory factor receptor α, nerve growth factor nerve growthfactor receptor, neuropoietin, neurotrophin-3, neurotrophin-4,oncostatin M (OSM), placenta growth factor, placenta growth factor 2,platelet-derived endothelial cell growth factor, platelet derived growthfactor, platelet derived growth factor A chain, platelet derived growthfactor AA, platelet derived growth factor AB, platelet derived growthfactor B chain, platelet derived growth factor BB, platelet derivedgrowth factor receptor α, platelet derived growth factor receptor β,pre-B cell growth stimulating factor, stem cell factor (SCF), stem cellfactor receptor, TNF, TNF0, TNF1, TNF2, transforming growth factor α,transforming growth factor β, transforming growth factor β1,transforming growth factor β1.2, transforming growth factor β2,transforming growth factor β3, transforming growth factor β5, latenttransforming growth factor β1, transforming growth factor β bindingprotein I, transforming growth factor β binding protein II, transforminggrowth factor β binding protein III, thymic stromal lymphopoietin(TSLP), tumor necrosis factor receptor type I, tumor necrosis factorreceptor type II, urokinase-type plasminogen activator receptor,phospholipase-activating protein (PUP), insulin, lectin ricin,prolactin, chorionic gonadotropin, follicle-stimulating hormone,thyroid-stimulating hormone, tissue plasminogen activator, IgG, IgE,IgM, IgA, and IgD, a-galactosidase, β-galactosidase, DNAse, fetuin,leutinizing hormone, estrogen, insulin, albumin, lipoproteins,fetoprotein, transferrin, thrombopoietin, urokinase, integrin, thrombin,leptin, adalimumab, denosumab, etanercept, or a protein in Table
 1. 6.The method according to claim 5 wherein a solution comprising an initialconcentration of the therapeutic protein between about 0.1 mg/ml andabout 5.0 mg/ml is adjusted to a pH value between about 5.0 and about8.0 prior to contacting with the activated water soluble polymer.
 7. Themethod of claim 5 comprising: a) a first step comprising adjusting thepH value of a solution comprising the therapeutic protein to a pH valuebetween about 5.0 and about 8.0, wherein the therapeutic proteinconcentration is between about 0.3 mg/ml and about 3.0 mg/ml; b) asecond step comprising oxidizing one or more carbohydrates on thetherapeutic protein, wherein the oxidizing agent is added to thesolution in the first step to result in a final concentration betweenabout 10 μM and about 1000 μM, under conditions comprising a time periodbetween about 0.1 minutes and about 5 hours; a temperature between about2° C. and about 37° C.; in the presence or absence of light, and with orwithout stirring; c) a third step comprising contacting the therapeuticprotein with a desired excess concentration of activated water solublepolymer, wherein the excess concentration is between about 1-molarexcess and about 300-molar excess, under conditions comprising a timeperiod between about 0.5 hours and about 24 hours, a temperature betweenabout 2° C. and about 37° C.; in the presence or absence of light; andwith or without stirring; d) a fourth step comprising adding anucleophilic catalyst to the solution of the third step, wherein thenucleophilic catalyst is added to result in a final concentrationbetween about 1 mM and about 50 mM, under conditions comprising a timeperiod between about 0.1 minutes and about 30 minutes; a temperaturebetween about 2° C. and about 37° C.; in the presence or absence oflight, and with or without stirring; e) a fifth step wherein thetherapeutic protein is incubated with the activated water solublepolymer and nucleophilic catalyst under conditions that allowconjugation of the activated water-soluble polymer to one or moreoxidized carbohydrates on the therapeutic protein, said conditionscomprising a time period between about 0.5 hours and about 24 hours, atemperature between about 2° C. and about 37° C.; in the presence orabsence of light, and with or without stirring; and f) a sixth stepwherein the conjugating the water soluble polymer to the one or moreoxidized carbohydrates of the therapeutic protein in the fifth step isstopped by the addition of a quenching agent selected from the groupconsisting of L-cysteine, methionine, glutathione, glycerol, Na₂S₂O₅(sodium meta bisulfite), tryptophane, tyrosine, histidine or derivativesthereof, kresol, imidazol, and combinations thereof; wherein thequenching agent is added to result in a final concentration of about 1mM and about 100 mM, under conditions comprising a time period betweenabout 5 minutes and about 120 minutes; a temperature between about 2° C.and about 37° C.; in the presence or absence of light, and with orwithout stirring.
 8. The method of claim 7 wherein the initialconcentration of the therapeutic protein in the first step is about 1mg/ml and the pH is about 6.0; wherein the final concentration ofoxidizing agent in the second step is about 400 μM, and the conditionsin the fifth step comprise a time period of about 10 minutes, atemperature of about 22° C., the absence of light and with stirring;wherein the excess concentration in the third step is about 50 molarexcess; wherein the conditions in the third step comprise a time periodof about 15 minutes, a temperature of about 22° C., the absence of lightand with stirring; wherein the final concentration of the nucleophiliccatalyst in the fourth step is about 10 mM, and the conditions in thefourth step comprise a time period of about 15 minutes, a temperature ofabout 22° C., the absence of light and with stirring; wherein theconditions of incubating the therapeutic protein with the activatedwater soluble polymer and nucleophilic catalyst in the fifth stepcomprise a time period of about 2 hours; a temperature of about 22° C.;the absence of light; and with stirring; and wherein the quenching agentin the sixth step is L-cysteine; and wherein the L-cysteine is added toresult in a final concentration of about 10 mM and the conditions in thesixth step comprise a time period of about 60 minutes, a temperature ofabout 22° C., the absence of light and with stirring.
 9. The methodaccording to claim 5 wherein the water soluble polymer is PSA.
 10. Themethod according to claim 5 wherein the water soluble polymer is PEG.11. The method according to claim 5 wherein the therapeutic protein isFIX.
 12. The method according to claim 5 wherein the therapeutic proteinis FVIIa.
 13. The method according to claim 5 wherein the therapeuticprotein is FVIII.
 14. The method according to claim 5 wherein the PSAcomprises an activated aminooxy linker selected from the groupconsisting of: a) a 3-oxa-pentane-1,5-dioxyarnine linker of the formula:

b) a 3,6,9-trioxa-undecane-1,11-dioxyamine linker of the formula:

c) a 3,6,9,12,15-penatoxa-heptadecane-1,17-dioxyamine linker of theformula:

wherein the PSA is oxidized by incubation with an oxidizing agent toform a terminal aldehyde group at the non-reducing end of the PSA. 15.The method according to claim 14 wherein the aminooxy linker is3-oxa-pentane-1,5-dioxyamine.